Binding molecules to the human OX40 receptor

ABSTRACT

The present disclosure provides isolated binding molecules that bind to the human OX40R, nucleic acid molecules encoding an amino acid sequence of the binding molecules, vectors comprising the nucleic acid molecules, host cells containing the vectors, methods of making the binding molecules, pharmaceutical compositions containing the binding molecules, and methods of using the binding molecules or compositions.

This application is a division of U.S. application Ser. No. 14/692,948filed Apr. 22, 2015, which is a division of U.S. application Ser. No.13/474,466 filed May 17, 2012 (now U.S. Pat. No. 9,028,824 issued May12, 2015), which is a division of U.S. application Ser. No. 13/099,196filed May 2, 2011 (now U.S. Pat. No. 8,236,930 issued Aug. 7, 2012),which is a division of U.S. application Ser. No. 12/332,944 filed Dec.11, 2008 (now U.S. Pat. No. 7,960,515 issued Jun. 14, 2011), whichclaims the benefit of U.S. Provisional Application No. 61/013,947 filedon Dec. 14, 2007, each of which is incorporated herein by reference inits entirety.

JOINT RESEARCH AGREEMENT

The disclosure and claims herein were made as a result of activitiesundertaken within the scope of a joint research agreement in effect onor before the date the claimed invention was made between Pfizer Inc.and Medarex, Inc.

BACKGROUND

The present disclosure relates to antibodies, and particularly toantibodies that bind to the OX40 receptor.

Enhancing anti-tumor T cell function represents a powerful and novelapproach for cancer treatment. Crucial components involved withgenerating an effective anti-tumor T cell response include enhancingCD4+ helper T cell activity to promote the generation of anti-tumorcytolytic T cells, and providing survival signals for memory andeffector T cells. A key receptor that has been shown to mediate theseresponses is the OX40 receptor. Sugamura, K., Ishii, N., Weinberg, A.Therapeutic targeting of the effector T-cell co-stimulatory moleculeOX40, Nature Rev. Imm. 4: 420-431 (2004); Hori, T. Roles of OX40 in thepathogenesis and control of diseases. Intn. J. Hematology. 83: 17-22(2006).

The OX40 receptor (OX40R) (also known as CD134, TNFRSF4, ACT-4, ACT35,and TXGP1L) is a member of the TNF receptor superfamily. The OX40R isfound to be expressed on activated CD4+ T-cells. High numbers of OX40R+T cells have been demonstrated within tumors (tumor infiltratinglymphocytes) and in the draining lymph nodes of cancer patients (Vetto,J. T. et al. 1997. Presence of the T-cell activation marker OX-40 ontumor infiltrating lymphocytes and draining lymph nodes cells frompatients with melanoma and head and neck cancers. Am. J. Surg. 174:258-265; Weinberg, A. D. et al. Engagement of the OX-40 receptor in vivoenhances antitumor immunity. J. Immunol. 164: 2160-69 (2000); Petty, J.K., et al. Survival in human colorectal cancer correlates withexpression of the T-cell costimulatory molecule OX-40 (CD134). Am. J.Surg. 183: 512-518 (2002)). It was shown in tumor models in mice thatengagement of the OX40R in vivo during tumor priming significantlydelayed and prevented the appearance of tumors as compared to controltreated mice (Weinberg et al., 2000). Therefore, it has beencontemplated to enhance the immune response of a mammal to an antigen byengaging the OX40R through the use of an OX40R binding agent (WO99/42585; Weinberg et al., 2000).

SUMMARY

The present disclosure provides isolated binding molecules that bind tothe human OX40R, including OX40R antibodies, antigen-binding fragmentsof the OX40R antibodies, and derivatives of the OX40R antibodies. Insome embodiments the binding molecule binds to the human OX40R with aK_(D) of 1×10⁻⁷ M or less and has agonist activity on the human OX40R.In some further embodiments, the binding molecule is a human monoclonalantibody that specifically binds to the human OX40R with a K_(D) of 100nM or less.

The present disclosure also provides a composition that comprises one ormore of the binding molecules and a pharmaceutically acceptable carrier.In some embodiments, the binding molecule is a human monoclonal OX40Rantibody or an antigen-binding fragment thereof. The composition mayfurther comprise additional pharmaceutical agents, such aschemotherapeutic agents, immunotherapeutic agents, and hormonaltherapeutic agents.

The present disclosure further provides therapeutic and diagnosticmethods using the binding molecules. In some embodiments, the disclosureprovides a method of treating or preventing cancer in a mammal,comprising administering to the mammal a therapeutically effectiveamount of a binding molecule or a composition comprising a bindingmolecule. In some other embodiments, the disclosure provides a method ofenhancing an immune response in a mammal, comprising administering tothe mammal a therapeutically effective amount of a binding molecule or acomposition comprising a binding molecule. In some particularembodiments the binding molecule used in the methods is a humanmonoclonal OX40R antibody or an antigen-binding fragment thereof.

The present disclosure further provides nucleic acid molecules thatencode an amino acid sequence of a binding molecule, vectors comprisingsuch nucleic acids, host cells comprising the vectors, and methods ofpreparing the binding molecules.

The disclosure also provides other aspects, which will be apparent fromthe entire disclosure, including the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1a and 1b are graphs showing that antibody 11D4 specifically bindsto the OX40R;

FIG. 2 is a graph showing the effect of cross-linked antibody 11D4 onOX40R-stimulated luciferase activity;

FIG. 3 is a graph showing the effect of antibody 11D4 on IL-2 productionby alloantigen primed T cells;

FIG. 4 is a graph showing the effect of antibody 11D4 on anti-CD3induced IL-2 production by primary T cells;

FIG. 5 is a graph showing the effect of antibody 11D4 on anti-CD3induced IL-2 production by cynomolgus primary T cells;

FIG. 6 shows the saturation binding curves with antibody 11D4 usingcynomolgus PBMC's from 14 donors stimulated with anti-CD3 and anti-CD28;

FIG. 7 shows the saturation binding curves with antibody 11D4 usinghuman PBMC's from 17 donors stimulated with anti-CD3 and anti-CD28;

FIG. 8 is a graph showing the effect of antibody 11D4 on the growth of Bcell lymphoma Raji in SCID mice;

FIG. 9 is a graph showing the effect of antibody 11D4 on the growth of Bcell lymphoma Raji 21 days after tumor injection;

FIG. 10 is a graph showing the effects of a single injection of antibody11D4 on the growth of the prostate tumor PC-3 in SCID mice;

FIG. 11 is a graph showing the effect of antibody 11D4 on the growth ofthe prostate tumor PC-3 in SCID mice 27 days after tumor injection;

FIG. 12 is a graph showing the effect of antibody 11D4 on the growth ofthe colon carcinoma LOVO in SCID mice;

FIG. 13 is a graph showing the effect of antibody 11D4 on the growth ofthe colon carcinoma LOVO in SCID mice 25 days after tumor injection;

FIG. 14 is a graph showing the effect of antibody 11D4 on the growth ofthe breast tumor BT474 in SCID mice; and

FIG. 15 is a graph showing the effect of antibody 11D4 on the growth ofthe breast tumor BT474 in SCID mice.

DETAILED DESCRIPTION

Definitions

The term “agonist” refers to a binding molecule, as defined herein,which upon binding to the OX40R, (1) stimulates or activates the OX40R,(2) enhances, increases, promotes, induces, or prolongs an activity,function, or presence of the OX40R, or (3) enhances, increases,promotes, or induces the expression of the OX40R.

The term “antibody” refers to an immunoglobulin molecule that istypically composed of two identical pairs of polypeptide chains, eachpair having one “light” (L) chain and one “heavy” (H) chain. Human lightchains are classified as kappa and lambda light chains. Heavy chains areclassified as mu, delta, gamma, alpha, or epsilon, and define theantibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Withinlight and heavy chains, the variable and constant regions are joined bya “J” region of about 12 or more amino acids, with the heavy chain alsoincluding a “D” region of about 3 or more amino acids. Each heavy chainis comprised of a heavy chain variable region (abbreviated herein asHCVR or V_(H)) and a heavy chain constant region. The heavy chainconstant region is comprised of three domains, C_(H)1, C_(H)2 andC_(H)3. Each light chain is comprised of a light chain variable region(abbreviated herein as LCVR or V_(L)) and a light chain constant region.The light chain constant region is comprised of one domain, CL. Theconstant regions of the antibodies may mediate the binding of theimmunoglobulin to host tissues or factors, including various cells ofthe immune system (e.g., effector cells) and the first component (C1q)of the classical complement system. The V_(H) and V_(L) regions can befurther subdivided into regions of hypervariability, termedcomplementarity determining regions (CDR), interspersed with regionsthat are more conserved, termed framework regions (FR). Each V_(H) andV_(L) is composed of three CDRs and four FRs, arranged fromamino-terminus to carboxy-terminus in the following order: FR1, CDR1,FR2, CDR2, FR3, CDR3, FR4. The variable regions of each heavy/lightchain pair (V_(H) and V_(L)), respectively, form the antibody bindingsite. The assignment of amino acids to each region or domain is inaccordance with the definitions of Kabat Sequences of Proteins ofImmunological Interest (National Institutes of Health, Bethesda, Md.(1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196:901-917;Chothia et al. (1989) Nature 342:878-883. The term “antibody”encompasses an antibody that is part of an antibody multimer (amultimeric form of antibodies), such as dimers, trimers, or higher-ordermultimers of monomeric antibodies. It also encompasses an antibody thatis linked or attached to, or otherwise physically or functionallyassociated with, a non-antibody moiety. Further, the term “antibody” isnot limited by any particular method of producing the antibody. Forexample, it includes, inter alia, recombinant antibodies, monoclonalantibodies, and polyclonal antibodies.

The term “antibody derivative” or “derivative” of an antibody refers toa molecule that is capable of binding to the same antigen (e.g., OX40R)that the antibody binds to and comprises an amino acid sequence of theantibody linked to an additional molecular entity. The amino acidsequence of the antibody that is contained in the antibody derivativemay be the full-length antibody, or may be any portion or portions of afull-length antibody. The additional molecular entity may be a chemicalor biological molecule. Examples of additional molecular entitiesinclude chemical groups, amino acids, peptides, proteins (such asenzymes, antibodies), and chemical compounds. The additional molecularentity may have any utility, such as for use as a detection agent,label, marker, pharmaceutical or therapeutic agent. The amino acidsequence of an antibody may be attached or linked to the additionalentity by chemical coupling, genetic fusion, noncovalent association orotherwise. The term “antibody derivative” also encompasses chimericantibodies, humanized antibodies, and molecules that are derived frommodifications of the amino acid sequences of an OX40R antibody, such asconservation amino acid substitutions, additions, and insertions.

The term “antigen-binding fragment” of an antibody refers to one or moreportions of a full-length antibody that retain the ability to bind tothe same antigen (e.g., OX40R) that the antibody binds to. The term“antigen-binding fragment” also encompasses the portion of an antibodythat is part of a larger molecule formed by covalent or noncovalentassociation of the antibody portion with one or more additionalmolecular entities. Examples of additional molecular entities includeamino acids, peptides, or proteins, such as the streptavidin coreregion, which may be used to make a tetrameric scFv molecule (Kipriyanovet al., (1995) Human Antibodies and Hybridomas 6:93-101), a cysteineresidue, a marker peptide, or a C-terminal polyhistidine tag, which maybe used to make bivalent and biotinylated scFv molecules (Kipriyanov etal., (1994) Mol. Immunol. 31:1047-1058).

The term “binding molecule” encompasses (1) antibody, (2)antigen-binding fragment of an antibody, and (3) derivative of anantibody, each as defined herein.

The term “binds to OX40R” or “binding to OX40R” refers to the binding ofa binding molecule, as defined herein, to the OX40R in an in vitroassay, such as a BIAcore assay. Binding means a binding affinity (K_(D))of 1×10⁻⁶ M or less.

The term “chimeric antibody” refers to an antibody that comprises aminoacid sequences derived from two or more different antibodies. The two ormore different antibodies may be from the same species or from two ormore different species.

The term “conservative amino acid substitution” refers to substitutionof an amino acid residue by another amino add residue, wherein the sidechain R groups of the two amino acid residues have similar chemicalproperties (e.g., charge or hydrophobicity). Examples of groups of aminoacids that have side chains with similar chemical properties include 1)aliphatic side chains: glycine, alanine, valine, leucine, andisoleucine; 2) aliphatic-hydroxyl side chains: serine and threonine; 3)amide-containing side chains: asparagine and glutamine; 4) aromatic sidechains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains:lysine, arginine, and histidine; 6) acidic side chains: aspartic acidand glutamic acid; and 7) sulfur-containing side chains: cysteine andmethionine. Conservative amino acid substitution groups can be, forexample, valine-leucine-isoleucine, phenylalanine-tyrosine,lysine-arginine, alanine-valine, glutamate-aspartate, andasparagine-glutamine.

The term “epitope” refers to the part of an antigen that is capable ofspecific binding to an antibody, or T-cell receptor, or otherwiseinteracting with a molecule. “Epitope” is also known in the art as“antigenic determinant.” An epitope generally consists of chemicallyactive surface groupings of molecules such as amino acids orcarbohydrate or sugar side chains and generally have specific threedimensional structural characteristics, as well as specific chargecharacteristics. An epitope may be “linear” or “conformational.” Once adesired epitope on an antigen is determined, antibodies to that epitopecan be generated, e.g., using the techniques described herein. Thegeneration and characterization of antibodies may also elucidateinformation about desirable epitopes. From this information, it is thenpossible to competitively screen antibodies for binding to the sameepitope. An approach to achieve this is to conduct cross-competitionstudies to find antibodies that competitively bind with one another,i.e., the antibodies compete for binding to the antigen. A highthroughput process for “binning” antibodies based upon theircross-competition is described in PCT Publication No. WO 03/48731.

The term “germline” refers to the nucleotide sequences of the antibodygenes and gene segments as they are passed from parents to offspring viathe germ cells. The germline sequence is distinguished from thenucleotide sequences encoding antibodies in mature B cells which havebeen altered by recombination and hypermutation events during the courseof B cell maturation.

The term “host cell” refers to a cell into which an expression vectorhas been introduced. The term encompasses not only the particularsubject cell but also the progeny of such a cell. Because certainmodifications may occur in succeeding generations due to either mutationor environmental influences, such progeny may not be identical to theparent cell, but are still included within the scope of the term “hostcell.” The term “human antibody” refers to an antibody consisting ofamino acid sequences of human immunoglobulin sequences only. A humanantibody may contain murine carbohydrate chains if produced in a mouse,in a mouse cell or in a hybridoma derived from a mouse cell. Humanantibodies may be prepared in a variety of ways known in the art.

The term “humanized antibody” refers to a chimeric antibody thatcontains amino acid residues derived from human antibody sequences. Ahumanized antibody may contain some or all of the CDRs from a non-humananimal antibody while the framework and constant regions of the antibodycontain amino acid residues derived from human antibody sequences.

The term “mammal” refers to any animal species of the Mammalia class.Examples of mammals include: humans; laboratory animals such as rats,mice, simians and guinea pigs; domestic animals such as cats, dogs,rabbits, cattle, sheep, goats, horses, and pigs; and captive wildanimals such as lions, tigers, elephants, and the like.

The term “isolated nucleic acid” refers to a nucleic acid molecule ofgenomic, cDNA, or synthetic origin, or a combination thereof, which isseparated from other nucleic acid molecules present in the naturalsource of the nucleic acid. For example, with regard to genomic DNA, theterm “isolated” includes nucleic acid molecules which are separated fromthe chromosome with which the genomic DNA is naturally associated.Preferably, an “isolated” nucleic acid is free of sequences whichnaturally flank the nucleic acid (i.e., sequences located at the 5′ and3′ ends of the nucleic acid of interest) in the genomic DNA of theorganism from which the nucleic acid is derived.

The term “isolated antibody” or “isolated binding molecule” refers to anantibody or a binding molecule that: (1) is not associated withnaturally associated components that accompany it in its native state;(2) is free of other proteins from the same species; (3) is expressed bya cell from a different species; or (4) does not occur in nature.Examples of isolated antibodies include an OX40R antibody that has beenaffinity purified using OX40R, an OX40R antibody that has been generatedby hybridomas or other cell line in vitro, and a human OX40R antibodyderived from a transgenic animal.

The term “K_(D)” refers to the equilibrium dissociation constant of aparticular antibody-antigen interaction and is used to describe thebinding affinity between a ligand (such as an antibody) and a protein(such as the OX40R). The smaller the equilibrium dissociation constant,the more tightly bound the ligand is, or the higher the affinity betweenligand and protein. A K_(D) can be measured by surface plasmonresonance, for example using the BIACORE™ system. An assay procedureusing the BIACORE™ system (BIAcore assay) is described in the Examplessection of this disclosure.

The term “off rate” or “kd” refers to the dissociation rate constant ofa particular antibody-antigen interaction. A dissociation rate constantcan be measured by surface plasmon resonance, for example using theBIACORE™.

The term “OX40R antibody” refers to an antibody, as defined herein,capable of binding to the human OX40R.

The terms “OX40 receptor” and “OX40R” are used interchangeably in thepresent application, and include the human OX40R, as well as variants,isoforms, and species homologs thereof. Accordingly, human bindingmolecules disclosed herein may, in certain cases, also bind to the OX40Rfrom species other than human. In other cases, the binding molecules maybe completely specific for the human OX40R and may not exhibit speciesor other types of cross-reactivity.

The term “specifically bind to the human OX40R” in reference to theinteraction of a binding molecule, e.g., an antibody, with its bindingpartner, e.g., an antigen, means that the K_(D) of a binding moleculefor binding to CD40, CD137, or CD271 is more than 100 fold the K_(D) forits binding to the human OX40R, as determined in an in vitro assay.

The term “vector” refers to a nucleic acid molecule capable oftransporting another nucleic acid molecule in a host cell. Examples ofvectors include plasmids, viral vectors, naked DNA or RNA expressionvectors, cosmid or phage vectors. Some vectors are capable of autonomousreplication in a host cell into which they are introduced (e.g.,bacterial vectors having a bacterial origin of replication and episomalmammalian vectors). Some vectors can be integrated into the genome of ahost cell upon introduction into the host cell, and thereby arereplicated along with the host genome (e.g., non-episomal mammalianvectors). Certain vectors are capable of directing the expression ofgenes to which they are operatively linked, and therefore may bereferred to as “expression vectors.”

As used herein, the twenty conventional amino acids and theirabbreviations follow conventional usage. See Immunology—A Synthesis(2^(nd) Edition, E. S. Golub and D. R. Gren, Eds., Sinauer Associates,Sunderland, Mass. (1991)).

Binding Molecules that Bind to the Human OX40R

The present disclosure provides isolated binding molecules that bind tothe human OX40R, including OX40R antibodies, antigen-binding fragmentsof the OX40R antibodies, and derivatives of the OX40R antibodies. Thebinding molecules are characterized by at least one of the followingfunctional properties: (a) bind to the human OX40R with a K_(D) of1×10⁻⁶ M or less; (b) have agonist activity on the human OX40R; (c) donot bind to CD40 receptor at concentration up to 500 nM; (d) do not bindto CD137 receptor at concentrations up to 500 nM; (e) do not bind toCD271 receptor at concentrations up to 500 nM; (f) are capable ofenhancing IL-2 production by isolated human T cells; (g) are capable ofenhancing immune response; (h) are capable of inhibiting tumor cellgrowth; and (i) have therapeutic effect on a cancer. In some embodimentsthe binding molecule binds to the human OX40R with a K_(D) of 1×10⁻⁷ Mor less, or 1×10⁻⁸ M or less, or 5×1×10⁻⁹ M or less.

Human OX40R Antibodies

In some first aspects, the present disclosure provides a human antibodythat binds to the human OX40R. In some embodiments, the human antibodyis a monoclonal antibody that specifically binds to the human OX40R witha K_(D) of 100 nM or less, preferably 10 nM or less, and has agonistactivity on the human OX40R. One example of such human antibodies is thehuman monoclonal antibody 11D4. The amino acid sequence of the heavychain and amino acid sequence of the variable region of the heavy chain(V_(H)) of antibody 11D4 are shown in SEQ ID NOs: 9 and 7, respectively.The amino acid sequence of the light chain and the amino acid sequenceof the variable region of the light chain (V_(L)) of antibody 11D4 areshown in SEQ ID NOS: 10 and 8, respectively. The isotypes of antibody11D4 are IgG2 for the heavy chain and Kappa for the light chain. Theallotypes of antibody 11D4 are G2(n−) for the heavy chain and Km3 forthe light chain. The mature heavy and light chain amino acid sequencesare derived from conceptual translation of DNA sequences in theexpression constructs. Antibody 11D4 contains no framework mutations inthe heavy chain or light chain, but contains one mutation in the heavychain CDR2.

Another illustrative antibody of the disclosure is the human monoclonalantibody 18D8. The amino acid sequence of the V_(H) region and V_(L)region of antibody 18D8 is shown in SEQ ID NOs: 19 and 20, respectively.The amino acid sequence of the heavy chain and light chain is shown inSEQ ID NOS: 21 and 22, respectively.

Given that 11D4 and 18D8 bind to the OX40R, the V_(H) and V_(L)sequences of each of them can be “mixed and matched” with other OX40Rantibodies to create additional antibodies. The binding of such “mixedand matched” antibodies to the OX40R can be tested using the bindingassays known in the art, including an assay described in the Examples.In one case, when V_(H) and V_(L) regions are mixed and matched, a V_(H)sequence from a particular V_(H)/V_(L) pairing is replaced with astructurally similar V_(H) sequence. Likewise, in another case a V_(L)sequence from a particular V_(H)/V_(L) pairing is replaced with astructurally similar V_(L) sequence.

Accordingly, in some embodiments, the disclosure provides an isolatedOX40R antibody that comprises: (1) a heavy chain variable region ofantibody 11D4 or 18D8, (2) a heavy chain variable region comprising anamino acid sequence of SEQ ID NOs: 7 or 19, or (3) a heavy chainvariable region comprising an amino acid sequence encoded by a nucleicacid sequence of SEQ ID NOs: 11 or 23. In some other embodiments, thedisclosure provides an isolated OX40R antibody that comprises: (1) alight chain variable region of antibody 11D4 or 18D8, (2) a light chainvariable region comprising an amino acid sequence of SEQ ID NOs: 8 or20, or (3) light chain variable region comprising an amino acid sequenceencoded by a nucleic acid sequence of SEQ ID NOs: 12 or 24.

In another aspect, the disclosure provides antibodies that comprise theCDR1, CDR2, and CDR3 of the heavy chain variable region (V_(H)) andCDR1, CDR2, and CDR3 of the light chain of 11D4 or 11D8. The amino acidsequence of the V_(H) CDR1, V_(H) CDR2, and V_(H) CDR3 of 11D4 is shownin SEQ ID NOs: 1, 2, and 3, respectively. The amino acid sequence of theV_(L) CDR1, V_(L) CDR2, and V_(L) CDR3 of antibody 11D4 is shown in SEQID NOs: 4, 5, and 6, respectively. The amino acid sequence of the V_(H)CDR1, V_(H) CDR2, and V_(H) CDR3 of antibody 18D8 is shown in SEQ IDNOs: 13, 14, and 15, respectively. The amino acid sequence of the V_(L)CDR1, V_(L) CDR2, and V_(L) CDR3 of antibody 18D8 is shown in SEQ IDNOs: 16, 17, and 18, respectively. The CDR regions are delineated usingthe Kabat system (Kabat, E. A., et al. (1991) Sequences of Proteins ofImmunological Interest, Fifth Edition, U.S. Department of Health andHuman Services, NIH Publication No. 91-3242).

Given that 11D4 and 18D8 bind to the human OX40R and thatantigen-binding specificity is provided primarily by the CDR1, CDR2, andCDR3 regions, the V_(H) CDR1, CDR2, and CDR3 sequences and V_(L) CDR1,CDR2, and CDR3 sequences can be “mixed and matched” to create additionalOX40R antibodies. For example, CDRs from different OX40R antibodies canbe mixed and matched, although each antibody will typically contain aV_(H) CDR1, CDR2, and CDR3 and a V_(L) CDR1, CDR2, and CDR3. The bindingof such “mixed and matched” antibodies to the OX40R can be tested usingthe binding assays described above and in the Examples (e.g., ELISAs,Biacore analysis). In one case, when V_(H) CDR sequences are mixed andmatched, the CDR1, CDR2 and/or CDR3 sequence from a particular V_(H)sequence is replaced with a structurally similar CDR sequence(s).Likewise, when V_(L) CDR sequences are mixed and matched, the CDR1, CDR2and/or CDR3 sequence from a particular V_(L) sequence typically isreplaced with a structurally similar CDR sequence(s). It will be readilyapparent to an ordinarily skilled artisan that novel V_(H) and V_(L)sequences can be created by substituting one or more V_(H) and/or V_(L)CDR region sequences with structurally similar sequences from the CDRsequences disclosed herein.

Accordingly, in some embodiments, the disclosure provides (1) anisolated monoclonal antibody that comprises at least one CDR selectedfrom V_(H) CDR1, V_(H) CDR2, or V_(H) CDR3 of antibody 11D4 or 18D8. Insome other embodiments, the disclosure provides an isolated monoclonalantibody that comprises at least one CDR selected from V_(L) CDR1, V_(L)CDR2 or V_(L) CDR3 of antibody 11D4 or 18D8. In some furtherembodiments, the disclosure provides an isolated monoclonal antibodythat comprises at least one CDR selected from: a V_(H) CDR1 comprisingthe amino acid sequence of SEQ ID NOs: 1 or 13, or a sequence thatdiffers from SEQ ID NOs: 1 or 3 by 1, 2, 3, or 4 conservative amino acidsubstitutions; a V_(H) CDR2 comprising the amino acid sequence of SEQ IDNOs: 2 or 14 or a sequence that differs from SEQ ID NOs: 2 or 14 by 1,2, 3, or 4 conservative amino acid substitutions; and a V_(H) CDR3comprising the amino acid sequence of SEQ ID NOs: 3 or 15 or a sequencethat differs from SEQ ID NOs: 3 or 15 by 1, 2, 3, or 4 conservativeamino acid substitutions.

In still some further embodiments, the disclosure provides an isolatedmonoclonal antibody that comprises at least one CDR selected from: aV_(L) CDR1 comprising the amino acid sequence of SEQ ID NOs: 4 or 16 ora sequence that differs from SEQ ID NOs: 4 or 16 by 1, 2, 3, or 4conservative amino acid substitutions; a V_(L) CDR2 comprising the aminoacid sequence of SEQ ID NOs: 5 or 17 or a sequence that differs from SEQID NOs: 5 or 17 by 1, 2, 3, or 4 conservative amino acid substitutions;and a V_(L) CDR3 comprising the amino acid sequence of SEQ ID NOs: 6 or18 or a sequence that differs from SEQ ID NOs: 6 or 18 by 1, 2, 3, or 4conservative amino acid substitutions.

In some cases, the C-terminal lysine of the heavy chain of an OX40Rantibody is cleaved (Harris R. J., J. of Chromotography, 705: 129-134(1995)). The heavy and/or light chain(s) of the OX40R antibodies mayoptionally include a signal sequence.

The class (e.g., IgG, IgM, IgE, IgA, or IgD) and subclass (e.g., IgG1,IgG2, IgG3, or IgG4) of the OX40R antibodies may be determined by anysuitable method. In general, the class and subclass of an antibody maybe determined using antibodies that are specific for a particular classand subclass of antibody. Such antibodies are commercially available.The class and subclass can be determined by ELISA, or Western Blot aswell as other techniques. Alternatively, the class and subclass may bedetermined by sequencing all or a portion of the constant domains of theheavy and/or light chains of the antibodies, comparing their amino acidsequences to the known amino acid sequences of various class andsubclasses of immunoglobulins, and determining the class and subclass ofthe antibodies. The OX40R antibodies can be an IgG, an IgM, an IgE, anIgA, or an IgD molecule. For example, the OX40R antibodies can be an IgGthat is an IgG1, IgG2, IgG3, or an IgG4 subclass. Thus, another aspectof the disclosure provides a method for converting the class or subclassof an OX40R antibody to another class or subclass. In some cases, anucleic acid molecule encoding a V_(L) or V_(H) that does not includesequences encoding C_(L) or C_(H) is isolated using methods well-knownin the art. The nucleic acid molecule then is operatively linked to anucleic acid sequence encoding a C_(L) or C_(H) from a desiredimmunoglobulin class or subclass. This can be achieved using a vector ornucleic acid molecule that comprises a C_(L) or C_(H) chain, asdescribed above. For example, an OX40R antibody that was originally IgMcan be class switched to an IgG. Further, the class switching may beused to convert one IgG subclass to another, e.g., from IgG1 to IgG2.Another method for producing an antibody comprising a desired isotypecomprises the steps of isolating a nucleic acid encoding a heavy chainof an OX40R antibody and a nucleic acid encoding a light chain of anOX40R antibody, isolating the sequence encoding the V_(H) region,ligating the V_(H) sequence to a sequence encoding a heavy chainconstant domain of the desired isotype, expressing the light chain geneand the heavy chain construct in a cell, and collecting the OX40Rantibody with the desired isotype.

Antigen-Binding Fragments

In another aspect, the present disclosure provides antigen-bindingfragments of any of the human OX40R antibodies as described hereinabove. In some embodiments, the antigen-binding fragment is selectedfrom: (1) a light chain of an OX40R antibody; (2) a heavy chain of anOX40R antibody; (3) a variable region from the light chain of an OX40Rantibody; (4) a variable region from the heavy chain of an OX40Rantibody; (5) one or more CDRs (two, three, four, five, or six CDRs) ofan OX40R antibody; or (6) three CDRs from the light chain and three CDRsfrom the heavy chain of an OX40R antibody. In some particularembodiments, the disclosure provides an antigen-binding fragment ofantibody 11D4 or 18D8. In some other particular embodiments, theantigen-binding fragments of an OX40R antibody include: (i) a Fabfragment, a monovalent fragment consisting of the V_(L), V_(H), C_(L)and C_(H)1 domains; (ii) a F(ab′)₂ fragment, a bivalent fragmentcomprising two Fab fragments linked by a disulfide bridge at the hingeregion; (iii) a Fd fragment consisting of the V_(H) and C_(H)1 domains;(iv) a Fv fragment consisting of the V_(L) and V_(H) domains of a singlearm of an antibody; (v) a dAb fragment (Ward et al., (1989) Nature341:544-546), which consists of a V_(H) domain; (vi) an isolated CDR,and (vii) single chain antibody (scFv), which is a polypeptidecomprising a V_(L) region of an antibody linked to a V_(H) region of anantibody. Bird et al., (1988) Science 242:423-426 and Huston et al.,(1988) Proc. Natl. Acad. Sci. USA 85:5879-5883. An antigen-bindingfragment may also comprise two or more shorter fragments, either fromthe same heavy drain or same light chain, or from different chains.Antigen-binding fragments, such as Fab and F(ab′)₂ fragments, can beprepared from whole antibodies using conventional techniques, such aspapain or pepsin digestion, respectively, of whole antibodies. They canalso be obtained using recombinant DNA techniques, as described herein.

Antibody Derivatives

In some further aspects, the present disclosure provides derivatives ofany of the OX40R antibodies as described herein above.

In one particular aspect, the antibody derivative is derived frommodifications of the amino acid sequences of 11D4 or 18D8. Amino acidsequences of any regions of the antibody chains may be modified, such asframework regions, CDR regions, or constant regions. The modificationscan be introduced by standard techniques known in the art, such assite-directed mutagenesis and random PCR-mediated mutagenesis, and maycomprise natural as well as non-natural amino acids.

Types of modifications include substitutions, insertions, deletions, orcombinations thereof, of one or more amino acids of an OX40R antibody.In some embodiments, the antibody derivative comprises 1, 2, 3, or 4amino acid substitutions in the heavy chain CDRs and/or one amino acidsubstitution in the light chain CDRs. In some embodiments, a derivativeof an OX40R antibody comprises one or more amino acid substitutionsrelative to the germline amino acid sequence of the human gene. In aparticular embodiment, one or more of those substitutions from germlineis in the CDR2 region of the heavy chain. In another particularembodiment, the amino acid substitutions relative to the germline are atone or more of the same positions as the substitutions relative togermline in antibodies 11D4 or 18D8. In another embodiment, the aminoacid substitution is to change one or more cysteines in an antibody toanother residue, such as, without limitation, alanine or serine. Thecysteine may be a canonical or non-canonical cysteine. The substitutioncan be made in a CDR or framework region of a variable domain or in theconstant domain of an antibody. Another type of amino acid substitutionis to eliminate asparagine-glycine pairs, which form potentialdeamidation sites, by altering one or both of the residues. In stillother embodiments, the amino acid substitution is a conservative aminoacid substitution. In one embodiment, the antibody derivative has 1, 2,3, or 4 conservative amino acid substitutions in the heavy chain CDRregions relative to the amino acid sequences of 11D4 or 18D8.

Another type of modification of an OX40R antibody is the alteration ofthe original glycosylation pattern of the antibody. The term“alteration” refers to deletion of one or more carbohydrate moietiesfound in the antibody, and/or adding one or more glycosylation sitesthat are not present in the antibody. Glycosylation of antibodies istypically N-linked. N-linked refers to the attachment of thecarbohydrate moiety to the side chain of an asparagine residue. Additionof glycosylation sites to the antibody is conveniently accomplished byaltering the amino acid sequence such that it contains one or more ofthe above-described tripeptide sequences (for N-linked glycosylationsites).

Still another type of modification involves removal of any carbohydratemoieties present on the antibody which may be accomplished chemically orenzymatically. Chemical deglycosylation requires exposure of theantibody to a compound, such as trifluoromethanesulfonic acid, or anequivalent compound. This treatment results in the cleavage of most orall sugars except the linking sugar (N-acetylglucosamine orN-acetylgalactosamine), while leaving the antibody intact. Chemicaldeglycosylation is described by Sojahr, H. T., and Bahl, O. P., Arch.Biochem. Biophys. 259 (1987) 52-57 and by Edge, A. S., et al. Anal.Biochem. 118 (1981) 131-137. Enzymatic cleavage of carbohydrate moietieson antibodies can be achieved by the use of a variety of endo- andexo-glycosidases as described by Thotakura, N. R., and Bahl, O. P.,Meth. Enzymol. 138 (1987) 350-359.

Examples of other modifications include acetylation, acylation,amidation, cross-linking, cyclization, disulfide bond formation,demethylation, formation of covalent cross-links, formation of cystine,formylation, hydroxylation, iodination, methylation, myristoylation,oxidation, pegylation, proteolytic processing, phosphorylation,prenylation, and sulfation.

In a further aspect, there is provided an antibody derivative thatcomprises an OX40R antibody, or antigen-binding fragment thereof, asdescribed herein, linked to an additional molecular entity. Examples ofadditional molecular entities include pharmaceutical agents, peptides orproteins, and detection agent or labels. Specific examples ofpharmaceutical agents that may be linked to an OX40R antibody includecytotoxic agents or other cancer therapeutic agents, and radioactiveisotopes. Specific examples of peptides or proteins that may be linkedto an OX40R antibody include antibodies, which may be the same OX40Rantibody or a different antibody. Specific examples of detection agentsor labels that may be linked to an OX40R antibody include (1)fluorescent compounds, such as fluorescein, fluorescein isothiocyanate,rhodamine, 5-dimethylamine-1-naphthalenesulfonyl chloride,phycoerythrin, and lanthanide phosphors; (2) enzymes, such ashorseradish peroxidase, β-galactosidase, luciferase, alkalinephosphatase, and glucose oxidase; (3) biotin; (4) a predeterminedpolypeptide epitope recognized by a secondary reporter, such as leucinezipper pair sequences, binding sites for secondary antibodies, metalbinding domains, and epitope tags. In a particular embodiment, theantibody derivative is an OX40R antibody multimer, which is a multimericform of an OX40R antibody, such as antibody dimers, trimers, orhigher-order multimers of monomeric antibodies. Individual monomerswithin an antibody multimer may be identical or different, i.e., theymay be heteromeric or homomeric antibody multimers. Individualantibodies within a multimer may have the same or different bindingspecificities. Multimerization of antibodies may be accomplished throughnatural aggregation of antibodies. For example, some percentage ofpurified antibody preparations (e.g., purified IgG1 molecules)spontaneously form protein aggregates containing antibody homodimers,and other higher-order antibody multimers. Alternatively, antibodyhomodimers may be formed through chemical linkage techniques known inthe art, such as through using heterobifunctional crosslinking agents.Suitable crosslinkers include those that are heterobifunctional, havingtwo distinctly reactive groups separated by an appropriate spacer (suchas m-maleimidobenzoyl-N-hydroxysuccinimide ester, succinimidyl4-(maleimidomethyl)cyclohexane-1-carboxylate, and N-succinimidylS-acethylthio-acetate) or homobifunctional (such as disuccinimidylsuberate). Such linkers are commercially available from Pierce ChemicalCompany, Rockford, Ill. Antibodies can also be made to multimerizethrough recombinant DNA techniques known in the art.

In still another aspect, the antibody derivative is a chimeric antibody,which comprises an amino acid sequence of a human OX40R antibodydescribed herein above. In one example, one or more CDRs from a humanOX40R antibody is combined with CDRs from an antibody from a non-humananimal, such as mouse or rat. In another example, all of the CDRs of thechimeric antibody are derived from human OX40R antibodies. In anotherexample, the CDRs from more than one human OX40R antibody are combinedin a chimeric antibody. Further, a chimeric antibody may comprise theframework regions derived from one human OX40R antibody and one or moreCDRs from one or more different human antibodies. Chimeric antibodiescan be generated using conventional methods known in the art. In someparticular embodiments, the chimeric antibody comprises one, two, orthree CDRs from the heavy chain variable region or from the light chainvariable region of an antibody selected from antibody 11D4 or 18D8.

Examples of other antibody derivatives provided by the presentdisclosure include single chain antibodies, diabodies, domainantibodies, nanobodies, and unibodies. A “single-chain antibody” (scFv)consists of a single polypeptide chain comprising a V_(L) domain linkedto a V_(H) domain wherein V_(L) domain and V_(H) domain are paired toform a monovalent molecule. Single chain antibody can be preparedaccording to method known in the art (see, for example, Bird et al.,(1988) Science 242:423-426 and Huston et al., (1988) Proc. Natl. Acad.Sci. USA 85:5879-5883). A “diabody” consists of two chains, each chaincomprising a heavy chain variable region connected to a light chainvariable region on the same polypeptide chain connected by a shortpeptide linker, wherein the two regions on the same chain do not pairwith each other but with complementary domains on the other chain toform a bispecific molecule. Methods of preparing diabodies are known inthe art (See, e.g., Holliger P. et al., (1993) Proc. Natl. Acad. Sci.USA 90:6444-6448, and Poljak R. J. et al., (1994) Structure2:1121-1123). Domain antibodies (dAbs) are small functional bindingunits of antibodies, corresponding to the variable regions of either theheavy or light chains of antibodies. Domain antibodies are wellexpressed in bacterial, yeast, and mammalian cell systems. Furtherdetails of domain antibodies and methods of production thereof are knownin the art (see, for example, U.S. Pat. Nos. 6,291,158; 6,582,915;6,593,081; 6,172,197; 6,696,245; European Patents 0368684 & 0616640;WO05/035572, WO04/101790, WO04/081026, WO04/058821, WO04/003019 andWO03/002609. Nanobodies are derived from the heavy chains of anantibody. A nanobody typically comprises a single variable domain andtwo constant domains (CH2 and CH3) and retains antigen-binding capacityof the original antibody. Nanobodies can be prepared by methods known inthe art (See e.g., U.S. Pat. No. 6,765,087, U.S. Pat. No. 6,838,254, WO06/079372). Unibodies consist of one light chain and one heavy chain ofa IgG4 antibody. Unibodies may be made by the removal of the hingeregion of IgG4 antibodies. Further details of unibodies and methods ofpreparing them may be found in WO2007/059782.

Methods of Producing the Binding Molecules

Binding molecules as disclosed herein can be produced by techniquesknown in the art, including conventional monoclonal antibodymethodology, e.g., the standard somatic cell hybridization technique ofKohler and Milstein (Nature 256: 495, (1975)), as well as othertechniques such as viral or oncogenic transformation of B lymphocytes.

Immunization of Non-Human Animals

The disclosure also provides a method for making OX40R antibodies orantigen-binding fragments thereof, which comprises immunizing anon-human animal that comprises human immunoglobulin loci with an OX40Rantigen, and isolating the antibody from the immunized animal or fromcells derived from the immunized animal.

Examples of suitable non-human animals include a transgenic ortranschromosomic animal, such as HuMAb Mouse®, KM Mouse®, “TC mice,” andXenomouse™. The HuMAb Mouse® (Medarex, Inc.) contains humanimmunoglobulin gene miniloci that encode unrearranged human heavy (μ andγ) and κ light chain immunoglobulin sequences, together with targetedmutations that inactivate the endogenous μ and κ chain loci (see e.g.,Lonberg, et al. (1994) Nature 368: 856-859). Accordingly, the miceexhibit reduced expression of mouse IgM or κ, and in response toimmunization, the introduced human heavy and light chain transgenesundergo class switching and somatic mutation to generate high affinityhuman IgGκ monoclonal antibodies (See, e.g., Harding, F. and Lonberg, N.(1995) Ann. N.Y. Acad. Sci. 764:536-546). Preparation and use of theHuMAb Mouse®, and the genomic modifications carried by such mice, iswell know in the art (See, e.g., Fishwild, D. et al. (1996) NatureBiotechnology 14: 845-851). The KM Mice™ carry a human heavy chaintransgene and a human light chain transchromosome and are described indetail in WO 02/43478. The Xenomouse™ (Abgenix, Inc.) contains largefragments of the human immunoglobulin loci and is deficient in mouseantibody production. This animal model is well known in the art (See,e.g., U.S. Pat. Nos. 5,939,598; 6,075,181; 6,114,598; 6,150,584; and6,162,963). “TC mice” are also engineered mice carrying both a humanheavy chain transchromosome and a human light chain transchromosome.Such mice are described in Tomizuka et al. (2000) Proc. Natl. Acad. Sci.USA 97:722-727.

The OX40R antigen for use to immunize the animal may be isolated and/orpurified OX40R and is preferably a human OX40R. In one embodiment, theOX40R antigen is a fragment of the human OX40R, preferably theextracellular domain of the OX40R. In another embodiment, the OX40Rantigen is a fragment that comprises at least one epitope of the humanOX40R. In another embodiment, the OX40R antigen is a cell that expressesOX40R on its cell surface, more particularly a cell that overexpressesthe OX40R on its cell surface. Immunization of the animals may be doneby any suitable method known in the art. (See, e.g., Harlow and Lane,Antibodies: A Laboratory Manual, New York: Cold Spring Harbor Press,1990). Particular methods for immunizing non-human animals such as mice,rats, sheep, goats, pigs, cattle and horses are well known in the art(See, e.g., Harlow and Lane (1990); U.S. Pat. No. 5,994,619). Example 1provides a method for immunizing HuMab mice.

After immunization of the animal with an OX40R antigen, antibodiesand/or antibody-producing cells can be obtained from the animal. In oneembodiment, serum is obtained from the animal and an immunoglobulinfraction may be obtained from the serum, or the OX40R antibodies may bepurified from the serum.

The OX40R antibodies may also be produced using antibody-producingimmortalized cells prepared from cells isolated from the immunizedanimal. After immunization, the lymph node and/or splenic B cells arecollected from the animal and immortalized by suitable means. Methods ofimmortalizing cells include, but are not limited to, transfecting themwith oncogenes, infecting them with an oncogenic virus and cultivatingthem under conditions that select for immortalized cells, subjectingthem to carcinogenic or mutating compounds, fusing them with animmortalized cell, e.g., a myeloma cell, and inactivating a tumorsuppressor gene (See, e.g., Harlow and Lane, supra). In a particularembodiment, the splenic B cells, collected from the immunized animal arefused to immortalized myeloma cells to form antibody-producingimmortalized hybridomas. The myeloma cells preferably do not secreteimmunoglobulin polypeptides (a non-secretory cell line). Immortalizedhybridomas are screened using the OX40 antigen (e.g., the OX40R, aportion thereof, or a cell expressing the OX40R). The initial screeningmay be performed, for example, using an enzyme-linked immunoassay(ELISA) or a radioimmunoassay. An example of ELISA screening isdescribed in WO 00/37504.

The OX40R antibody-producing cells, e.g., hybridomas, are selected,cloned, and further screened for desirable characteristics, includingrobust growth, high antibody production, and desirable antibodycharacteristics, as discussed further below. Hybridomas can be expandedin vivo in syngeneic animals, in animals that lack an immune system,e.g., nude mice, or in cell culture in vitro.

Thus, methods are provided for producing a cell that produces a humanmonoclonal OX40R antibody or an antigen-binding fragment thereof,comprising: (a) immunizing a non-human transgenic animal with an OX40Rantigen; (b) allowing the animal to mount an immune response to theOX40R antigen; (c) isolating antibody-producing cells from the animal;and (d) immortalizing the antibody-producing cells. In one embodiment,the method further comprises (e) creating individual monoclonalpopulations of the immortalized antibody-producing cells; and (f)screening the immortalized antibody-producing cells that produce adesired OX40R antibody.

Nucleic Acids, Vectors, Host Cells, and Recombinant Methods of ProducingOX40R Antibodies

Another aspect of the disclosure provides an isolated nucleic acidmolecule encoding an amino acid sequence of a binding molecule thatbinds the human OX40R. The amino acid sequence encoded by the nucleicacid molecule may be any portion of an intact antibody, such as a CDR, asequence comprising one, two, or three CDRs, or a variable region of aheavy chain or light chain, or may be a full-length heavy chain or lightchain. In some embodiments, the nucleic acid molecule encodes an aminoacid sequence that comprises (1) a CDR3 region, particularly a heavychain CDR3 region, of antibodies 11D4 or 18D8; (2) a variable region ofa heavy chain or variable region of a light chain of antibodies 11D4 or18D8; or (3) a heavy chain or a light chain of antibodies 11D4 or 18D8.In other embodiments, the nucleic acid molecule encodes a polypeptidethat comprises an amino acid sequence selected from the group consistingof SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 13, 14, 15, 16, 17, 18,19, 20, 21, and 22. In still other embodiments, the nucleic acidmolecule is selected from the group consisting of SEQ ID NOs: 11, 12,23, and 24.

The nucleic acid molecules provided by the disclosure may be obtainedfrom any source that produces an OX40R antibody. mRNA from OX40Rantibody-producing cells may be isolated by standard techniques, clonedand/or amplified using PCR and library construction techniques, andscreened using standard protocols to obtain nucleic acid moleculesencoding an amino acid sequence of an OX40R antibody. The mRNA may beused to produce cDNA for use in the polymerase chain reaction (PCR) orcDNA cloning of antibody genes. In one embodiment, the nucleic acidmolecule is obtained from a hybridoma that expresses an OX40R antibody,as described above, preferably a hybridoma that has as one of its fusionpartners a non-human transgenic animal cell that expresses humanimmunoglobulin genes. In another embodiment, the hybridoma is derivedfrom a non-human, non-transgenic animal.

A nucleic acid molecule encoding the heavy chain of an OX40R antibodymay be constructed by fusing a nucleic acid molecule encoding the heavyvariable region with a nucleic acid molecule encoding a constant regionof a heavy chain. Similarly, a nucleic acid molecule encoding the lightchain of an OX40R antibody may be constructed by fusing a nucleic acidmolecule encoding the light chain variable region with a nucleic acidmolecule encoding a constant region of a light chain. The nucleic acidmolecules encoding the VH and VL chain may be converted to full-lengthantibody genes by inserting them into expression vectors alreadyencoding heavy chain constant and light chain constant regions,respectively, such that the VH segment is operatively linked to theheavy chain constant region (CH) segment(s) within the vector and the VLsegment is operatively linked to the light chain constant region (CL)segment within the vector. Alternatively, the nucleic acid moleculesencoding the VH or VL chains are converted into full-length antibodygenes by linking, e.g., ligating, the nucleic acid molecule encoding aVH chain to a nucleic acid molecule encoding a CH chain using standardmolecular biological techniques. The same may be achieved using nucleicacid molecules encoding VL and CL chains. The sequences of human heavyand light chain constant region genes are known in the art. See, e.g.,Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed.,NIH Publ. No. 91-3242, 1991. Nucleic acid molecules encoding thefull-length heavy and/or light chains may then be expressed from a cellinto which they have been introduced and the OX40R antibody isolated.

The nucleic acid molecules may be used to recombinantly express largequantities of OX40R antibodies, as described below. The nucleic acidmolecules may also be used to produce other binding molecules providedby the disclosure, such as chimeric antibodies, single chain antibodies,immunoadhesins, diabodies, mutated antibodies, and antibody derivatives,as described elsewhere herein. In one embodiment, a nucleic acidmolecule is used as probe or PCR primer for specific antibody sequences.For instance, a nucleic acid molecule probe may be used in diagnosticmethods or a nucleic acid molecule PCR primer may be used to amplifyregions of DNA that could be used, inter alia, to isolate nucleic acidsequences for use in producing variable regions of the OX40R antibodies.

Once DNA molecules encoding the V_(H) and V_(L) segments of an OX40Rantibody are obtained, these DNA molecules can be further manipulated byrecombinant DNA techniques, for example to convert the variable regiongenes to full-length antibody chain genes, to Fab fragment genes, or toa scFv gene. In these manipulations, a V_(L)- or V_(H)-encoding DNAmolecule is operatively linked to another DNA molecule encoding anotherpolypeptide, such as an antibody constant region or a flexible linker.The term “operatively linked,” as used in this context, means that thetwo DNA molecules are joined such that the amino acid sequences encodedby the two DNA molecules remain in-frame.

The isolated DNA molecule encoding the V_(H) region can be converted toa full-length heavy chain gene by operatively linking the V_(H)-encodingDNA molecule to another DNA molecule encoding heavy chain constantregions (CH1, CH2 and CH3). The sequences of human heavy chain constantregion genes are known in the art (see, e.g., Kabat, E. A., et al.(1991) Sequences of Proteins of Immunological Interest, Fifth Edition,U.S. Department of Health and Human Services, NIH Publication No.91-3242) and DNA fragments encompassing these regions can be obtained bystandard PCR amplification. The heavy chain constant region can be anIgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region, but mostpreferably is an IgG1 or IgG2 constant region. The IgG1 constant regionsequence can be any of the various alleles or allotypes known to occuramong different individuals, such as Gm(1), Gm(2), Gm(3), and Gm(17).These allotypes represent naturally occurring amino acid substitutionsin the IgG1 constant regions. For a Fab fragment heavy chain gene, theV_(H)-encoding DNA can be operatively linked to another DNA moleculeencoding only the heavy chain CH1 constant region. The CH1 heavy chainconstant region may be derived from any of the heavy chain genes.

The isolated DNA molecule encoding the V_(L) region can be converted toa full-length light chain gene (as well as a Fab light chain gene) byoperatively linking the V_(L)-encoding DNA molecule to another DNAmolecule encoding the light chain constant region, C_(L). The sequencesof human light chain constant region genes are known in the art (seee.g., Kabat, E. A., et al. (1991) Sequences of Proteins of ImmunologicalInterest, Fifth Edition, U.S. Department of Health and Human Services,NIH Publication No. 91-3242) and DNA fragments encompassing theseregions can be obtained by standard PCR amplification. The light chainconstant region can be a kappa or lambda constant region. The kappaconstant region may be any of the various alleles known to occur amongdifferent individuals, such as Inv(1), Inv(2), and Inv(3). The lambdaconstant region may be derived from any of the three lambda genes.

To create a scFv gene, the V_(H)- and V_(L)-encoding DNA fragments areoperatively linked to another fragment encoding a flexible linker, e.g.,encoding the amino acid sequence (Gly₄-Ser)₃, such that the V_(H) andV_(L) sequences can be expressed as a contiguous single-chain protein,with the V_(L) and V_(H) regions joined by the flexible linker (Seee.g., Bird et al., (1988) Science 242:423-426; Huston et al., (1988)Proc. Natl. Acad. Sci. USA 85:5879-5883; McCafferty et al., (1990)Nature 348:552-554). The single chain antibody may be monovalent, ifonly a single V_(H) and V_(L) are used, bivalent, if two V_(H) and V_(L)are used, or polyvalent, if more than two V_(H) and V_(L) are used.Bispecific or polyvalent antibodies may be generated that bindspecifically to OX40R and to another molecule.

In another aspect, the present disclosure provides a vector, whichcomprises a nucleic acid molecule described herein above. The nucleicacid molecule may encode a portion of a light chain or heavy chain (suchas a CDR or a variable region), a full-length light or heavy chain,polypeptide that comprises a portion or full-length of a heavy or lightchain, or an amino acid sequence of an antibody derivative or antigenbinding fragment. To express a binding molecule, a DNA molecule encodingpartial or full-length binding molecule is inserted into an expressionvector such that the DNA molecule is operatively linked totranscriptional and translational control sequences. In this context,the term “operatively linked” is intended to mean that the DNA moleculeis ligated into a vector such that transcriptional and translationalcontrol sequences within the vector serve their intended function ofregulating the transcription and translation of the DNA molecule. Theexpression vector and expression control sequences are chosen to becompatible with the expression host cell used. Expression vectorsinclude, for example, plasmids, retroviruses, adenoviruses,adeno-associated viruses (AAV), plant viruses such as cauliflower mosaicvirus, tobacco mosaic virus, cosmids, YACs, and EBV derived episomes.The DNA molecule encoding an amino acid sequence of the light chain andDNA molecule encoding an amino acid sequence of the heavy chain can beinserted into separate vectors or in the same vector. The DNA moleculeis inserted into the expression vector by any suitable methods (e.g.,ligation of complementary restriction sites on the antibody genefragment and vector, or blunt end ligation if no restriction sites arepresent).

An example of a suitable expression vector is one that encodes afunctionally complete human C_(H) or C_(L) immunoglobulin sequence, withappropriate restriction sites engineered so that any V_(H) or V_(L)sequence can be inserted and expressed. The expression vector also canencode a signal peptide that facilitates secretion of the amino acidsequence of the antibody chain from a host cell. The DNA encoding theamino acid sequence of an antibody chain may be cloned into the vectorsuch that the signal peptide is linked in-frame to the amino terminus ofthe amino acid sequence of the antibody chain. The signal peptide can bean immunoglobulin signal peptide or a heterologous signal peptide (i.e.,a signal peptide from a non-immunoglobulin protein).

In addition to the nucleic acid sequence encoding an amino acid sequenceof an OX40R antibody (antibody chain genes), the expression vectorscarry regulatory sequences that control the expression of the antibodychain genes in a host cell. The design of the expression vector,including the selection of regulatory sequences, may depend on suchfactors as the choice of the host cell to be transformed, the level ofexpression of protein desired, and so forth. Regulatory sequences formammalian host cell expression include viral elements that direct highlevels of protein expression in mammalian cells, such as promotersand/or enhancers derived from retroviral LTRs, cytomegalovirus (CMV)(such as the CMV promoter/enhancer), Simian Virus 40 (SV40) (such as theSV40 promoter/enhancer), adenovirus, (e.g., the adenovirus major latepromoter (AdMLP)), polyoma and strong mammalian promoters such as nativeimmunoglobulin and actin promoters. For further description of viralregulatory elements, and sequences thereof, see e.g., U.S. Pat. Nos.5,168,062, 4,510,245, and 4,968,615.

In addition to the antibody chain nucleic acid sequences and regulatorysequences, the recombinant expression vectors may carry additionalsequences, such as sequences that regulate replication of the vector inhost cells and selectable marker genes. The selectable marker genefacilitates selection of host cells into which the vector has beenintroduced (see e.g., U.S. Pat. Nos. 4,399,216, 4,634,665 and5,179,017). Selectable marker genes include the dihydrofolate reductase(DHFR) gene (for use in dhfr-host cells with methotrexateselection/amplification), the neomycin phosphotransferase gene (for G418selection), and the glutamate synthetase gene. The design of theexpression vector, including the selection of regulatory sequences, maydepend on a number of factors, such as the choice of the host cell to betransformed, the level of expression of protein desired, and so forth.Nucleic acid molecules encoding binding molecules and vectors comprisingthese nucleic acid molecules can be used for transformation of asuitable host cell for recombinant production of a binding molecule. Asuitable host cell is transformed with one or more expression vectorscarrying nucleic acid molecules encoding an amino acid sequence of abinding molecule such that the amino acid sequence is expressed in thehost cell and, typically, secreted into the medium in which the hostcell is cultured and from which medium the amino acid sequence can berecovered. Transformation of host cells can be by carried out by anysuitable method know in the art, such as those disclosed in U.S. Pat.Nos. 4,399,216, 4,912,040, 4,740,461, and 4,959,455.

The host cell may be a mammalian, insect, plant, bacterial, or yeastcell. Examples of mammalian cell lines suitable as host cells includeChinese hamster ovary (CHO) cells, NSO cells, SP2 cells, HEK-293T cells,NIH-3T3 cells, HeLa cells, baby hamster kidney (BHK) cells, Africangreen monkey kidney cells (COS), human hepatocellular carcinoma cells(e.g., Hep G2), A549 cells, and a number of other cell lines. Examplesof insect cell lines include Sf9 or Sf21 cells. Examples of plant hostcells include Nicotiana, Arabidopsis, duckweed, corn, wheat, potato, andso forth. Bacterial host cells include E. coli and Streptomyces species.Examples of yeast host cells include Schizosaccharomyces pombe,Saccharomyces cerevisiae, and Pichia pastoris.

Amino acid sequences of a binding molecule expressed by different celllines or in transgenic animals may have different glycosylation.However, all binding molecules encoded by the nucleic acid moleculesprovided herein, or comprising the amino acid sequences provided hereinare part of the present invention, regardless of the glycosylation ofthe binding molecules.

In another aspect, the present disclosure provides a method forproducing an OX40R antibody or antigen-binding fragment thereof usingphage display. The method comprises (a) synthesizing a library of humanantibodies on phage, (b) screening the library with the OX40R or aportion thereof, (c) isolating phage that binds the OX40R or a portionthereof, and (d) obtaining the antibody from the phage. One exemplarymethod for preparing the library of antibodies comprises the step of:(a) immunizing a non-human animal comprising human immunoglobulin lociwith OX40R or an antigenic portion thereof to create an immune response;(b) extracting antibody-producing cells from the immunized animal; (c)isolating RNA encoding heavy and light chains of the OX40R antibodiesfrom the extracted cells; (d) reverse transcribing the RNA to producecDNA; (e), amplifying the cDNA; and (f) inserting the cDNA into a phagedisplay vector such that antibodies are expressed on the phage.Recombinant human OX40R antibodies or antigen binding fragments thereofcan be isolated by screening a recombinant combinatorial antibodylibrary. The library may be a scFv phage display library, generatedusing human V_(L) and V_(H) cDNAs prepared from mRNA isolated from Bcells. Methods for preparing and screening such libraries are known inthe art. Kits for generating phage display libraries are commerciallyavailable (e.g., the Pharmacia Recombinant Phage Antibody System,catalog no. 27-9400-01; and the Stratagene SurfZAP™ phage display kit,catalog no. 240612).

In one case, to isolate and produce human OX40R antibodies with thedesired characteristics, a human OX40R antibody as described herein isfirst used to select human heavy and light chain sequences havingsimilar binding activity toward OX40R using methods known in the art,such as the epitope imprinting methods described in WO 93/06213. Theantibody libraries used in this method may be scFv libraries preparedand screened as described in WO 92/01047, McCafferty et al., Nature348:552-554 (1990); and Griffiths et al., EMBO J. 12:725-734 (1993). ThescFv antibody libraries may be screened using human CCR2 as the antigen.

Once initial human V_(L) and V_(H) regions are selected, “mix and match”experiments are performed, in which different pairs of the initiallyselected V_(L) and V_(H) segments are screened for OX40R binding toselect V_(L)/V_(H) pair combinations. Additionally, to further improvethe quality of the antibody, the V_(L) and V_(H) segments of theV_(L)/V_(H) pair(s) can be randomly mutated, within the CDR3 region ofV_(H) and/or V_(L), in a process analogous to the in vivo somaticmutation process responsible for affinity maturation of antibodiesduring a natural immune response. This in vitro affinity maturation canbe accomplished by amplifying V_(H) and V_(L) domains using PCR primerscomplimentary to the V_(H) CDR3 or V_(L) CDR3, respectively, whichprimers have been “spiked” with a random mixture of the four nucleotidebases at certain positions such that the resultant PCR products encodeV_(H) and V_(L) segments into which random mutations have beenintroduced into the V_(H) and/or V_(L) CDR3 regions. These randomlymutated V_(H) and V_(L) segments can be re-screened for binding toOX40R.

Following screening and isolation of an OX40R antibody or antigenbinding portion from a recombinant immunoglobulin display library,nucleic acids encoding the selected binding molecule can be recoveredfrom the display package (e.g., from the phage genome) and subclonedinto other expression vectors by recombinant DNA techniques. If desired,the nucleic acid can further be manipulated to create other antibodyforms, as described below. To express a recombinant human antibodyisolated by screening of a combinatorial library, the DNA encoding theantibody is cloned into a recombinant expression vector and introducedinto mammalian host cells, as described above.

Pharmaceutical Compositions

In another aspect, the present disclosure provides a composition, e.g.,a pharmaceutical composition, containing one or a combination of bindingmolecules provided by the disclosure, and optionally a pharmaceuticallyacceptable carrier. The compositions can be prepared by conventionalmethods known in the art.

In some embodiments, the composition comprises an OX40R antibody or anantigen-binding fragment thereof. In a particular embodiment, thecomposition comprises antibody 11D4 or antibody 18D8, or aantigen-binding fragment of either antibody. In still other embodiments,the composition comprises a derivative of antibody 11D4 or antibody18D8.

The term “pharmaceutically acceptable carrier” refers to any inactivesubstance that is suitable for use in a formulation for the delivery ofa binding molecule. A carrier may be an antiadherent, binder, coating,disintegrant, filler or diluent, preservative (such as antioxidant,antibacterial, or antifungal agent), sweetener, absorption delayingagent, wetting agent, emulsifying agent, buffer, and the like. Examplesof suitable pharmaceutically acceptable carriers include water, ethanol,polyols (such as glycerol, propylene glycol, polyethylene glycol, andthe like) dextrose, vegetable oils (such as olive oil), saline, buffer,buffered saline, and isotonic agents such as sugars, polyalcohols,sorbitol, and sodium chloride.

The compositions may be in any suitable forms, such as liquid,semi-solid, and solid dosage forms. Examples of liquid dosage formsinclude solution (e.g., injectable and infusible solutions),microemulsion, liposome, dispersion, or suspension. Examples of soliddosage forms include tablet, pill, capsule, microcapsule, and powder. Aparticular form of the composition suitable for delivering a bindingmolecule is a sterile liquid, such as a solution, suspension, ordispersion, for injection or infusion. Sterile solutions can be preparedby incorporating the antibody in the required amount in an appropriatecarrier, followed by sterilization microfiltration. Generally,dispersions are prepared by incorporating the antibody into a sterilevehicle that contains a basic dispersion medium and other carriers. Inthe case of sterile powders for the preparation of sterile liquid,methods of preparation include vacuum drying and freeze-drying(lyophilization) to yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof. The various dosage forms of the compositions can beprepared by conventional techniques known in the art.

The relative amount of a binding molecule included in the compositionwill vary depending upon a number of factors, such as the specificbinding molecule and carriers used, dosage form, and desired release andpharmacodynamic characteristics. The amount of a binding molecule in asingle dosage form will generally be that amount which produces atherapeutic effect, but may also be a lesser amount. Generally, tinsamount will range from about 0.01 percent to about 99 percent, fromabout 0.1 percent to about 70 percent, or from about 1 percent to about30 percent relative to the total weight of the dosage form.

In addition to the binding molecule, one or more additional therapeuticagents may be included in the composition. Examples of the additionaltherapeutic agents are described herein below. The suitable amount ofthe additional therapeutic agent to be included in the composition canbe readily selected by a person skilled in the art, and will varydepending on a number of factors, such as the particular agent andcarriers used, dosage form, and desired release and pharmacodynamiccharacteristics. The amount of the additional therapeutic agent includedin a single dosage form will generally be that amount of the agent whichproduces a therapeutic effect, but may be a lesser amount as well.

Use of the Binding Molecules and Pharmaceutical Compositions

Binding molecules and pharmaceutical compositions comprising a bindingmolecule provided by the present disclosure are useful for therapeutic,diagnostic, or other purposes, such as enhancing an immune response,treating cancer, enhancing efficacy of other cancer therapy, orenhancing vaccine efficacy, and have a number of utilities, such as foruse as medicaments or diagnostic agents. Thus, in another aspect, thepresent disclosure provides methods of using the binding molecules orpharmaceutical compositions.

In one particular aspect, methods are provided for enhancing immuneresponse in a mammal, comprising administering to the mammal atherapeutically effective amount of a binding molecule provided by thedisclosure. In some embodiments, the binding molecule is an OX40Rantibody or antigen-binding fragment thereof and the mammal is a human.In a further embodiment, the binding molecule is antibody 11D4 orantibody 18D8, or an antigen-binding fragment of either antibody. Theterm “enhancing immune response” or its grammatical variations, meansstimulating, evoking, increasing, improving, or augmenting any responseof a mammal's immune system. The immune response may be a cellularresponse (i.e. cell-mediated, such as cytotoxic T lymphocyte mediated)or a humoral response (i.e. antibody mediated response), and may be aprimary or secondary immune response. Examples of enhancement of immuneresponse include increased CD4+ helper T cell activity and generation ofcytolytic T cells. The enhancement of immune response can be assessedusing a number of in vitro or in vivo measurements known to thoseskilled in the art, including, but not limited to, cytotoxic Tlymphocyte assays, release of cytokines (for example IL-2 production),regression of tumors, survival of tumor bearing animals, antibodyproduction, immune cell proliferation, expression of cell surfacemarkers, and cytotoxicity. Typically, methods of the disclosure enhancethe immune response by a mammal when compared to the immune response byan untreated mammal or an animal not treated using the claimed methods.In one embodiment, the method enhances a cellular immune response,particularly a cytotoxic T cell response. In another embodiment, thecellular immune response is a T helper cell response. In still anotherembodiment, the immune response is a cytokine production, particularlyIL-2 production.

In another particular aspect, the present disclosure provides a methodof treating cancer in a mammal, comprising administering to the mammal atherapeutically effective amount of a binding molecule provided by thedisclosure. The term “treating cancer” or “treatment of cancer” refersto causing a desirable or beneficial effect in a mammal diagnosed with acancer. The desirable or beneficial effect may include inhibition offurther growth or spread of cancer cells, death of cancer cells,inhibition of reoccurrence of cancer, reduction of pain associated withthe cancer, or improved survival of the animal. Inhibition ofreoccurrence of cancer contemplates cancer sites and surrounding tissuewhich have previously been treated by radiation, chemotherapy, surgery,or other techniques. The effect can be either subjective or objective.For example, if the animal is human, the human may note improved vigoror vitality or decreased pain as subjective symptoms of improvement orresponse to therapy. Alternatively, the clinician may notice a decreasein tumor size or tumor burden based on physical exam, laboratoryparameters, tumor markers or radiographic findings. Some laboratorysigns that the clinician may observe for response to treatment includenormalization of tests, such as white blood cell count, red blood cellcount, platelet count, erythrocyte sedimentation rate, and variousenzyme levels. Additionally, the clinician may observe a decrease in adetectable tumor marker. Alternatively, other tests can be used toevaluate objective improvement, such as sonograms, nuclear magneticresonance testing and positron emissions testing. In some embodiments,the binding molecule is an OX40R antibody or an antigen-binding fragmentthereof provided by the disclosure. In a further embodiment the bindingmolecule is antibody 11D4 or 18D8, or an antigen-binding fragment ofeither antibody. In a further embodiment, the mammal is a human.

In another particular aspect, the present disclosure provides a methodof preventing cancer in a mammal, comprising administering to the mammala therapeutically effective amount of a binding molecule provided by thedisclosure. The term “preventing cancer” or “prevention of cancer”refers to delaying, inhibiting, or preventing the onset of a cancer in amammal in which the onset of oncogenesis or tumorigenesis is notevidenced but a predisposition for cancer is identified whetherdetermined by genetic screening, for example, or otherwise. The termalso encompasses treating a mammal having premalignant conditions tostop the progression of, or cause regression of, the premalignantconditions towards malignancy. Examples of premalignant conditionsinclude hyperplasia, dysplasia, and metaplasia. In some embodiments, thebinding molecule is an OX40R antibody or a fragment thereof provided bythe disclosure. In a further embodiment the binding molecule is antibody11D4 or 18D8, or an antigen-binding fragment of either antibody. In afurther embodiment, the mammal is a human.

A variety of cancers, whether malignant or benign and whether primary orsecondary, may be treated or prevented with a method provided by thedisclosure. Examples of such cancers include lung cancers such asbronchogenic carcinoma (e.g., squamous cell carcinoma, small cellcarcinoma, large cell carcinoma, and adenocarcinoma), alveolar cellcarcinoma, bronchial adenoma, chondromatous hamartoma (noncancerous),and sarcoma (cancerous); heart cancer such as myxoma, fibromas, andrhabdomyomas; bone cancers such as osteochondromas, condromas,chondroblastomas, chondromyxoid fibromas, osteoid osteomas, giant celltumors, chondrosarcoma, multiple myeloma, osteosarcoma, fibrosarcomas,malignant fibrous histiocytomas, Ewing's tumor (Ewing's sarcoma), andreticulum cell sarcoma; brain cancer such as gliomas (e.g., glioblastomamultiforme), anaplastic astrocytomas, astrocytomas, oligodendrogliomas,medulloblastomas, chordoma, Schwannomas, ependymomas, meningiomas,pituitary adenoma, pinealoma, osteomas, hemangioblastomas,craniopharyngiomas, chordomas, germinomas, teratomas, dermoid cysts, andangiomas; cancers in digestive system such as leiomyoma, epidermoidcarcinoma, adenocarcinoma, leiomyosarcoma, stomach adenocarcinomas,intestinal lipomas, intestinal neurofibromas, intestinal fibromas,polyps in large intestine, and colorectal cancers; liver cancers such ashepatocellular adenomas, hemangioma, hepatocellular carcinoma,fibrolamellar carcinoma, cholangiocarcinoma, hepatoblastoma, andangiosarcoma; kidney cancers such as kidney adenocarcinoma, renal cellcarcinoma, hypernephroma, and transitional cell carcinoma of the renalpelvis; bladder cancers; hematological cancers such as acute lymphocytic(lymphoblastic) leukemia, acute myeloid (myelocytic, myelogenous,myeloblastic, myelomonocytic) leukemia, chronic lymphocytic leukemia(e.g., Sezary syndrome and hairy cell leukemia), chronic myelocytic(myeloid, myelogenous, granulocytic) leukemia, Hodgkin's lymphoma,non-Hodgkin's lymphoma, B cell lymphoma, mycosis fungoides, andmyeloproliferative disorders (including myeloproliferative disorderssuch as polycythemia vera, myelofibrosis, thrombocythemia, and chronicmyelocytic leukemia); skin cancers such as basal cell carcinoma,squamous cell carcinoma, melanoma, Kaposi's sarcoma, and Paget'sdisease; head and neck cancers; eye-related cancers such asretinoblastoma and intraoccular melanocarcinoma; male reproductivesystem cancers such as benign prostatic hyperplasia, prostate cancer,and testicular cancers (e.g., seminoma, teratoma, embryonal carcinoma,and choriocarcinoma); breast cancer; female reproductive system cancerssuch as uterine cancer (endometrial carcinoma), cervical cancer(cervical carcinoma), cancer of the ovaries (ovarian carcinoma), vulvarcarcinoma, vaginal carcinoma, fallopian tube cancer, and hydatidiformmole; thyroid cancer (including papillary, follicular, anaplastic, ormedullary cancer); pheochromocytomas (adrenal gland); noncancerousgrowths of the parathyroid glands; pancreatic cancers; and hematologicalcancers such as leukemias, myelomas, non-Hodgkin's lymphomas, andHodgkin's lymphomas.

In practicing the therapeutic methods, the binding molecules may beadministered alone us monotherapy, or administered in combination withone or more additional therapeutic agents or therapies. Thus, in anotheraspect, the present disclosure provides a combination therapy, whichcomprises a binding molecule provided by the disclosure in combinationwith one or more additional therapies or therapeutic agents. The term“additional therapy” refers to a therapy which does not employ a bindingmolecule provided by the disclosure as a therapeutic agent. The term“additional therapeutic agent” refers to any therapeutic agent otherthan a binding molecule provided by the disclosure. In some embodiments,the binding molecule is antibody 11D4 or 18D8, or an antigen-bindingfragment of either antibody. In one particular aspect, the presentdisclosure provides a combination therapy for treating cancer in amammal, which comprises administering to the mammal a therapeuticallyeffective amount of a binding molecule provided by the disclosure incombination with one or more additional therapeutic agents. In a furtherembodiment, the mammal is a human.

A wide variety of cancer therapeutic agents may be used in combinationwith a binding molecule. One of ordinary skill in the art will recognizethe presence and development of other cancer therapies which can be usedin combination with the methods and binding molecules of the presentdisclosure, and will not be restricted to those forms of therapy setforth herein. Examples of categories of additional therapeutic agentsthat may be used in the combination therapy for treating cancer include(1) chemotherapeutic agents, (2) immunotherapeutic agents, and (3)hormone therapeutic agents.

The term “chemotherapeutic agent” refers to a chemical or biologicalsubstance that can cause death of cancer cells, or interfere withgrowth, division, repair, and/or function of cancer cells. Examples ofchemotherapeutic agents include those that are disclosed in WO2006/088639, WO 2006/129163, and US 20060153808, the disclosures ofwhich are incorporated herein by reference. Examples of particularchemotherapeutic agents include: (1) alkylating agents, such aschlorambucil (LEUKERAN), mcyclophosphamide (CYTOXAN), ifosfamide (IFEX),mechlorethamine hydrochloride (MUSTARGEN), thiotepa (THIOPLEX),streptozotocin (ZANOSAR), carmustine (BICNU, GLIADEL WAFER), lomustine(CEENU), and dacarbazine (DTIC-DOME); (2) alkaloids or plant vincaalkaloids, including cytotoxic antibiotics, such as doxorubicin(ADRIAMYCIN), epirubicin (ELLENCE, PHARMORUBICIN), daunorubicin(CERUBIDINE, DAUNOXOME), nemorubicin, idarubicin (IDAMYCIN PFS,ZAVEDOS), mitoxantrone (DHAD, NOVANTRONE), dactinomycin (actinomycin D,COSMEGEN), plicamycin (MITHRACIN), mitomycin (MUTAMYCIN), and bleomycin(BLENOXANE), vinorelbine tartrate (NAVELBINE)), vinblastine (VELBAN),vincristine (ONCOVIN), and vindesine (ELDISINE); (3) antimetabolites,such as capecitabine (XELODA), cytarabine (CYTOSAR-U), fludarabine(FLUDARA), gemcitabine (GEMZAR), hydroxyurea (HYDRA), methotrexate(FOLEX, MEXATE, TREXALL), nelarabine (ARRANON), trimetrexate(NEUTREXIN), and pemetrexed (ALIMTA); (4) Pyrimidine antagonists, suchas 5-fluorouracil (5-FU); capecitabine (XELODA), raltitrexed (TOMUDEX),tegafur-uracil (UFTORAL), and gemcitabine (GEMZAR); (5) taxanes, such asdocetaxel (TAXOTERE), paclitaxel (TAXOL); (6) platinum drugs, such ascisplatin (PLATINOL) and carboplatin (PARAPLATIN), and oxaliplatin(ELOXATIN); (7) topoisomerase inhibitors, such as irinotecan(CAMPTOSAR), topotecan (HYCAMTIN), etoposidc (ETOPOPHOS, VEPESSID,TOPOSAR), and teniposide (VUMON); (8) epipodophyllotoxins(podophyllotoxin derivatives), such as etoposide (ETOPOPHOS, VEPESSID,TOPOSAR); (9) folic acid derivatives, such as leucovorin (WELLCOVORIN);(10) nitrosoureas, such as carmustine (BiCNU), lomustine (CeeNU); (11)inhibitors of receptor tyrosine kinase, including epidermal growthfactor receptor (EGFR), vascular endothelial growth factor (VEGF),insulin receptor, insulin-like growth factor receptor (IGFR), hepatocytegrowth factor receptor (HGFR), and platelet-derived growth factorreceptor (PDGFR), such as gefitinib (IRESSA), erlotinib (TARCEVA),bortezomib (VELCADE), imatinib mesylate (GLEEVEC), genefitinib,lapatinib, sorafenib, thalidomide, sunitinib (SUTENT), axitinib,rituximab, trastuzumab (HERCEPTIN), cetuximab (ERBITUX), bevacizumab(AVASTIN), and ranibizumab (LUCENTIS), lym-1 (ONCOLYM), antibodies toinsulin-like growth factor-1 receptor (IGF-1R) that are disclosed inWO2002/053596); (12) angiogenesis inhibitors, such as bevacizumab(AVASTIN), suramin (GERMANIN), angiostatin, SU5416, thalidomide, andmatrix metalloproteinase inhibitors (such as batimastat and marimastat),and those that are disclosed in WO2002055106; and (13) proteasomeinhibitors, such as bortezomib (VELCADE).

The term “immunotherapeutic agents” refers to a chemical or biologicalsubstance that can enhance an immune response of a mammal. Examples ofimmunotherapeutic agents include: bacillus Calmette-Guerin (BCG);cytokines such as interferons; vaccines such as MyVax personalizedimmunotherapy; Onyvax-P, Oncophage, GRNVAC1, FavId, Provenge, GVAX,Lovaxin C, BiovaxID, GMXX, and NeuVax; and antibodies such asalemtuzumab (CAMPATH), bevacizumab (AVASTIN), cetuximab (ERBITUX),gemtuzunab ozogamicin (MYLOTARG), ibritumomab tiuxetan (ZEVALIN),panitumumab (VECTIBIX), rituximab (RITUXAN, MABTHERA), trastuzumab(HERCEPTIN), tositumomab (BEXXAR), tremelimumab, CAT-3888, and agonistantibodies to CD40 receptor that are disclosed in WO2003/040170.

The term “hormone therapeutic agent” refers to a chemical or biologicalsubstance that inhibits or eliminates the production of a hormone, orinhibits or counteracts the effect of a hormone on the growth and/orsurvival of cancerous cells. Examples of such agents suitable for themethods herein include those that are disclosed in US20070117809.Examples of particular hormone therapeutic agents include tamoxifen(NOLVADEX), toremifene (Fareston), fulvestrant (FASLODEX), anastrozole(ARIMIDEX), exemestane (AROMASIN), letrozole (FEMARA), megestrol acetate(MEGACE), goserelin (ZOLADEX), and leuprolide (LUPRON). The bindingmolecules of this disclosure may also be used in combination withnon-drug hormone therapies such as (1) surgical methods that remove allor part of the organs or glands which participate in the production ofthe hormone, such as the ovaries, the testicles, the adrenal gland, andthe pituitary gland, and (2) radiation treatment, in which the organs orglands of the patient are subjected to radiation in an amount sufficientto inhibit or eliminate the production of the targeted hormone.

The combination therapy for treating cancer also encompasses thecombination of a binding molecule provided by the disclosure withsurgery to remove a tumor. The binding molecule may be administered tothe mammal before, during, or after the surgery.

The combination therapy for treating cancer also encompasses combinationof a binding molecule provided by the disclosure with radiation therapy,such as ionizing (electromagnetic) radiotherapy (e.g., X-rays or gammarays) and particle beam radiation therapy (e.g., high linear energyradiation). The source of radiation can be external or internal to themammal. The binding molecule may be administered to the mammal before,during, or after the radiation therapy.

Administration of the Binding Molecules and Compositions

The binding molecules and compositions provided by the presentdisclosure can be administered via any suitable enteral route orparenteral route of administration. The term “enteral route” ofadministration refers to the administration via any part of thegastrointestinal tract. Examples of enteral routes include oral,mucosal, buccal, and rectal route, or intragastric route. “Parenteralroute” of administration refers to a route of administration other thanenteral route. Examples of parenteral routes of administration includeintravenous, intramuscular, intradermal, intraperitoneal, intratumor,intravesical, intraarterial, intrathecal, intracapsular, intraorbital,intracardiac, transtracheal, intraarticular, subcapsular, subarachnoid,intraspinal, epidural and intrasternal, subcutaneous, or topicaladministration. The antibodies and compositions of die disclosure can beadministered using any suitable method, such as by oral ingestion,nasogastric tube, gastrostomy tube, injection, infusion, implantableinfusion pump, and osmotic pump. The suitable route and method ofadministration may vary depending on a number of factors such as thespecific antibody being used, the rate of absorption desired, specificformulation or dosage form used, type or severity of the disorder beingtreated, the specific site of action, and conditions of the patient, andcan be readily selected by a person skilled in the art

The term “therapeutically effective amount” of a binding molecule refersto an amount that is effective for an intended therapeutic purpose. Forexample, in the context of enhancing an immune response, a“therapeutically effective amount” is any amount that is effective instimulating, evoking, increasing, improving, or augmenting any responseof a mammal's immune system. In the context of treating cancer, a“therapeutically effective amount” is any amount that is sufficient tocause any desirable or beneficial effect in the mammal being treated,such as inhibition of further growth or spread of cancer cells, death ofcancer cells, inhibition of reoccurrence of cancer, reduction of painassociated with the cancer, or improved survival of the mammal. In amethod of preventing cancer, a “therapeutically effective amount” is anyamount that is effective in delaying, inhibiting, or preventing theonset of a cancer in the mammal to which the binding molecule isadministered. The therapeutically effective amount of a binding moleculeusually ranges from about 0.001 to about 500 mg/kg, and more usuallyabout 0.05 to about 100 mg/kg, of the body weight of the mammal. Forexample, the amount can be about 0.3 mg/kg, 1 mg/kg, 3 mg/kg, 5 mg/kg,10 mg/kg, 50 mg/kg, or 100 mg/kg of body weight of the mammal. In someembodiments, the therapeutically effective amount of an OX40R antibodyis in the range of about 0.1-30 mg/kg of body weight of the mammal. Theprecise dosage level to be administered can be readily determined by aperson skilled in the art and will depend on a number of factors, suchas the type, and severity of the disorder to be treated, the particularbinding molecule employed, the route of administration, the time ofadministration, the duration of the treatment, the particular additionaltherapy employed, the age, sex, weight, condition, general health andprior medical history of the patient being treated, and like factorswell known in the medical arts.

A binding molecule or composition is usually administered on multipleoccasions. Intervals between single doses can be, for example, weekly,monthly, every three months or yearly. An exemplary treatment regimenentails administration once per week, once every two weeks, once everythree weeks, once every four weeks, once a month, once every 3 months oronce every three to 6 months. Typical dosage regimens for an OX40Rantibody include 1 mg/kg body weight or 3 mg/kg body weight viaintravenous administration, using one of the following dosing schedules:(i) every four weeks for six dosages, then every three months; (ii)every three weeks; (iii) 3 mg/kg body weight once followed by 1 mg/kgbody weight every three weeks.

EXAMPLES Example 1: Preparation of OX40R Antibodies

Illustrative antibodies in accordance with the disclosure were prepared,selected, and assayed as follows:

Immunization with the OX40R Antigen and Selection of Mice ProducingOX40R Monoclonal Antibodies:

Fully human monoclonal antibodies to human OX40R were prepared usinghuman Ig transgenic mouse strains HCo7, HCo12, Hco17, and Hco27 as wellas the human transchromosomal/transgenic strain, KM (Medarex, Inc.).These strains all express fully human antibodies that areindistinguishable from antibodies isolated from humans.

In the transgenic strains, both the endogenous mouse kappa light chaingene and the endogenous mouse heavy chain gene were homozygouslydisrupted as described in Chen et al. (1993) EMBO J. 12:821-830 and inExample 1 of WO 01/09187, respectively. Moreover, they carry a humankappa light chain transgene, KCo5, as described in Fishwild et al.(1996) Nature Biotechnology 14:845-851. In contrast, the transgenicstrains are distinct with respect to their human heavy chain genes. TheHCo7 strain carries the HCo7 human heavy chain transgene as described inU.S. Pat. Nos. 5,545,806, 5,625,825, and 5,545,807; the HCo12 straincarries the HCo12 human heavy chain transgene as described in Example 2of WO 01/09187; the Hco17 strain carries the Hco17 human heavy chaintransgene as described in Example 8 of Deshpande et al., US2005/0191293A1; the Hco27 strain carries the Hco27 human heavy chaintransgene as described in Example 5 of PCT/US2008/072640 filed 8 Aug.2008. The KM strain carries a human mini-chromosome as described inIshida et al., (2002), Cloning and Stem Cells, 4: 91-102.

General immunization schemes for HuMab mice are described in Lonberg etal. (1994) Nature 368(6474): 856-859; Fishwild et al. (1996) NatureBiotechnology 14: 845-851; and PCT Publication WO 98/24884.

HuMab mice of the HCo7, HCo12, Hco17, Hco27 and KM strains wereimmunized beginning at 6-16 weeks of age with 15-25 μgs of purifiedhuman recombinant OX40R-Ig protein and murine pre-B cell line, 300-19(Reth, M. G. et al., Nature 312 29: 418-42, 1984; Alt, F. et al., Cell27: 381-390, 1981), transfected to express human OX40R in Ribi adjuvant.The purified human recombinant OX40R-Ig protein is a construct of theextracellular domain (amino acids 1-220) of human OX40R fused to theconstant region of human IgG1. Administration was via injectionintra-peritoneally, subcutaneously or into the footpad at 3-28 dayintervals, up to a total of 10 immunizations. Immune response wasmonitored via ELISA and FACS screening as described below.

Selection of HuMab Mice Producing OX40R Antibodies:

To select HuMab mice producing antibodies that bind to the OX40R, bloodfrom the immunized mice was obtained and analyzed by ELISA for specificbinding to purified human OX40R recombinant protein, and by FACS forbinding to a cell line expressing full length human OX40R, and not to acontrol cell line not expressing OX40R.

ELISA binding assay was as described by Fishwild et al. (1996), NatureBiotechnology 14: 845-851. Briefly, microtiter plates were coated using50 μl/well of a purified recombinant OX40R-Ig solution containing 1μg/ml in PBS, and incubated overnight at 4° C. The wells were thenblocked using 200 μl/well of 5% chicken serum in PBS/Tween (0.05%).Dilutions of plasma from OX40R-immunized mice were added to each welland incubated for 1 hour at ambient temperature. The plates were washedwith PBS/Tween and then incubated with a goat-anti-human IgG Fcpolyclonal antibody conjugated with horseradish peroxidase (HRP) for 1hour at room temperature. After washing, the plates were developed withABTS substrate (Moss Inc., product #: ABTS-1000 mg/ml) and analyzed byspectrophotometer at OD 405.

FACS assay was carried out according to conventional procedures.Briefly, OX40R-expressing 300-19 cells were incubated with serum fromimmunized mice diluted at 1:20. Cells were washed and specific antibodybinding was detected with FITC-labeled anti-human IgG Ab. Flowcytometric analyses were performed on a FACS flow cytometry instrument(Becton Dickinson, San Jose, Calif.).

Mice that developed the highest titers of OX40R antibodies were used forfusions. Fusions were performed as described below and hybridomasupernatants were tested for anti-OX40R activity by ELISA and FACS.

Generation of Hybridomas Producing Human Monoclonal Antibodies to OX40R:

The mice selected above were boosted intravenously with OX40R-Ig at 3days and then again at 2 days prior to sacrifice and removal of thespleen and/or lymph nodes.

The splenocytes and/or lymph node lymphocytes isolated from theimmunized HuMab or KM mice were fused to SP2/0 non-secreting mousemyeloma cells (ATCC, CRL-1581) using electrofusion (E-fusion, CytoPulse™ technology, Cyto Pulse™ Sciences, Inc., Glen Burnie, Md.),according to standard or manufacturer-recommended protocols. Briefly,single cell suspensions of splenocytes and/or lymph node lymphocytesfrom immunized mice were prepared and then combined with an equal numberof SP2/0 non-secreting mouse myeloma cells; E-fusion was then performed.

The cells were then plated at 2×10⁴ cells/well in flat bottom microtiterplate, and incubated for 10-14 days in selective medium containing 10%fetal bovine serum, 10% P388D1 (ATCC, CRL-TIB-63) conditioned medium,3-5% (IGEN) in DMEM (Mediatech, Herndon, Va., Cat. No. CRL 10013, withhigh glucose, L-glutamine and sodium pyruvate), 7 mM HEPES, 0.055 mM2-mercaptoethanol, 0.1 IU/mL penicillin-0.1 mg/mL streptomycin, and1×HAT (Sigma, Cat. No. CRL-P-7185).

After 1-2 weeks, cells were cultured in medium in which the HAT wasreplaced with HT. Approximately 10-14 days after cell plating,supernatants from individual wells were screened for the presence ofhuman gamma, kappa antibodies. The supernatants which scored positivefor human gamma, kappa were then screened by ELISA and FACS (using theprotocol described above) for human OX40R monoclonal IgG antibodies. Theantibody-secreting hybridomas were transferred to 24 well plates,screened again and, if confirmed positive for human OX40R IgG monoclonalantibodies, were subcloned at least twice by limiting dilution. Thestable subclones were then cultured in vitro to generate small amountsof antibody in tissue culture medium for further characterization.

Example 2: Biological/Pharmacological Examples

A. In Vitro Study Procedures:

Binding to the Extracellular Domain of the OX40R: A human OX40-Ig fusionprotein was diluted in BupH™ Carbonate buffer, pH 9.4 (Pierce, Rockford,Ill.) was coated onto 96-well Maxisorb plates (Nunc, Roskilde, Denmark)at 100 μl/well (0.25 μg/ml) and incubated overnight at 4° C. The Plateswere washed three times with wash buffer containing 0.05% Tween 20(Sigma, St Louis, Mo.) diluted in PBS (Sigma, St Louis, Mo.) and blockedwith 300 μl/well of 0.5% BSA (Sigma, St Louis, Mo.) in PBS for 1 hour atRT °. Next, the plates were washed and incubated with anti-human OX40reactive antibodies diluted in blocking buffer at various concentrations(100 μl/well) and incubated for 1 hour at RT °. The plates were thenwashed and incubated for one hour at RT ° with a horse radish peroxidaselabeled anti-human kappa chain antibody (Bethyl Laboratories,Montgomery, Tex.) at 25 ng/ml in blocking buffer. Finally, the assayplates were washed and 100 μl/well of 1-Step Turbo-TMB substrate(Pierce, Rockford, Ill.) was added for 30 minutes at RT °. The reactionwas stopped by adding an equal volume of 2M H₂SO₄ and absorbance wasread at 450 nm on a Molecular Devices Spectra Max 340 (MolecularDevices, Sunnyvale, Calif.).

FACS Based Binding to Cell Surface OX40R:

OX40R-expressing cell lines (see below) or activated primary peripheralblood mononuclear cells (see below) were used to assess binding on boththe human and cynomolgus OX40 receptors. Cells were harvested and washed(5×10⁵/tube) using wash buffer at RT °. The wash buffer consisted ofPBS, 2% heat-inactivated fetal bovine serum (Hyclone, Logan, Utah) and0.02% sodium azide (Sigma, St. Louis, Mo.). Next, 100 μl of variousconcentrations of antibody was added to the cells (starting at 30 ug/mland using a 3-fold titration) diluted in wash buffer containing 0.005mg/ml of cytocholasin B (Sigma, St. Louis, Mo.). The cells were gentlyrocked at RT ° for 3 hours. Next, the cells were washed twice andresuspended in 0.5 ml/tube with cold wash buffer and 10,000 events werecollected and analyzed using a Becton Dickinson FACSCalibur andCellQuest software (San Jose, Calif.).

Biacore Assay:

The Biosensor biospecific interaction analysis instrument (BIAcore 2000)uses surface plasmon resonance to measure molecular interactions on aCM5 sensor chip. Changes in the refractive indices between two media,glass and carboxymethylated dextran, caused by the interaction ofmolecules to the dextran side of the sensor chip, is measured andreported as changes in arbitrary reflectance units (RU) as detailed inthe manufacturer's application notes.

The carboxymethylated dextran surfaces on a CM5 sensor chip wereactivated by derivatization with 0.05 M N-hydroxysuccinimide mediated by0.2 M N-ethyl-N′-(dimethylaminopropyl) carbodiimide for 7 min.Streptavidin (Sigma S-4762) at a concentration of 500 μg/ml, in 10 mM Naacetate, pH 4.5, was injected onto three surfaces (Flow Cell-2, 3 and 4)at a rate of 5 μl/min and covalently immobilized to the flow cellsurfaces with approximately 2500 RU's. 35 μl of 10 mM Na acetate bufferwas injected over Flow cell-1 during immobilization in place of antigento make an activated blank surface to measure non-specific binding.Deactivation of unreacted N-hydroxysuccinimide esters on all four Flowcells was performed using 1M ethanolamine hydrochloride, pH 8.5.Following immobilization, the flow cells are cleaned of any unreacted orpoorly bound material with 5 regeneration injections of 5 μl of 50 mMNaOH until a stable baseline was achieved.

Biotinylated CD134-muIg (Ancell 513-030), at a concentration of 10 μg/mlat a flow rate of 5 μl/min was manually injected over Flow cells-2, 3and 4 to achieve 3 surface densities: Fc-2=150 RU, Fc-3=375 RU andFc-4=580 RU. The different density surfaces were prepared to monitor thepossibility of mass transport limited binding during association phaseand rebinding during dissociation, both artifacts that are influenced bysurface density that must be avoided.

A dilution series of the OX40R antibodies were prepared over aconcentration range of 666 nM to 66 pM by half logs in running buffer(0.01M HEPES, pH 7.4, 0.15M NaCl, 3 mM EDTA, 0.005% polysorbate 20(v/v)). The flow rate was set at 5 μl/min and 25 μl of eachconcentration point sample was injected over the sensor chip with aregeneration injection of 5 μl of 50 mM NaOH between each concentrationof antibody injected. Dissociation time was 5 min. The data was analyzedusing BIAevaluation 3.0 global fit software (separate analysis of eachconcentration point).

Epitope Characterization:

300-19 cells expressing a recombinant human OX40-CD40 fusion constructcorresponding to 1-235 amino acid sequence of OX40 (extracellular andtransmembrane domain) and 216-278 amino acid sequence of CD40(intracellular domain) was used for antibody epitope analysis. TheOX40-CD40 expressing cell line was grown in RPMI medium (Gibco, GrandIsland, N.Y.) supplemented with 10% fetal calf serum (Hyclone, Logan,Utah), 10 mM hepes, 1% penicillin-streptomycin, 2 mM L-glutamine, 0.1 mMnon-essential amino acids and 0.05 mM 2-mercapthoethanol (Gibco, GrandIsland, N.Y.). 300-19.hCD134.2 cells (5×10⁵/tube) were washed once in 3mls of cold wash buffer (PBS, 2% FBS and 0.02% sodium azide). The cellsupernatant was aspirated and 100 μl of wash buffer containing 300 μg/mlof primary unconjugated OX40 reactive antibody was added to the cellpellet, mixed and incubated for 30 minutes at 4° C. Next, a fluorochromelabeled secondary antibody was added to the tube, mixed and incubatedfor an additional 30 minutes at 4° C. The OX40 reactive fluorochromelabeled antibodies included either 10 μl of phycoerythrin (PE) labeledBer Act 35 (Caltag Laboratories, Burlingame, Calif.), PE-labeled L106(BD Pharmingen, San Jose, Calif.) or Alexa Fluor 647 conjugated OX40Rantibody. The OX40R antibody was labeled with fluorochrome using theAlex Fluor 647 protein labeling kit as described by the manufacturer(Molecular Probes, Eugene, Oreg.). After staining, cells were thenwashed 3 times with wash buffer, resuspended in cold wash buffer and10,000 events were collected and analyzed using a Becton DickinsonFACSCalibur and CellQuest software (San Jose, Calif.). Antibodies weredemeaned as binding to the same epitope when the primary antibodyblocked the staining of the secondary fluorochrome labeled antibody bymore than 80%.

Antibody OX40 Ligand-OX40R Inhibition Assay:

Antibodies were tested for their ability to block the binding of the300-19 human-OX40 ligand (L) expressing cells to OX40-human IgG1 fusionprotein coated plates. The 300-19-OX40L cell line was grown in RPMImedium (Gibco, Grand Island, N.Y.) supplemented with 10% fetal bovineserum (Hyclone, Logan, Utah), 10 mM HEPES, 1% penicillin-streptomycin, 2mM L-glutamine, 0.1 mM non-essential amino acids, 0.05 mM2-mercapthoethanol and 0.5 mg/ml Geneticin (Gibco, Grand Island, N.Y.).The OX40-human IgG1 fusion protein contains the first 220 amino acids ofthe extracellular OX40 protein. The fusion protein was coated onto toNunc Maxisorb plates (Nunc, Roskilde, Denmark) in 100μ/well (5 μg/ml) incoating buffer (BupH, Carbonate-Bicarbonate buffer, Pierce, Rockford,Ill.) and incubated overnight at 4° C. Next, plates were blotted on apaper towel to remove fluid, blocked with 200 μl/well with blockingbuffer (5% Carnation Milk diluted in PBS) and incubated at RT ° for twohours. Plates were washed with PBS and various dilutions of antibodydiluted in PBS were then added (50 μl/well) to the assay plate andincubated at RT ° for 30 minutes. Next, 50 μl/well of cells in PBS at6×10⁵/well were added to the antibody containing wells and incubated foran additional 60 minutes at 37° C. in a 5% CO₂ humidified chamber. Theplates were gently washed 2 times with PBS to remove non-adherent cellsand cell activity in the wells was measured by adding 200 μl of a 20ug/ml Fluorescein Diacetate (Sigma, St. Louis, Mo.) PBS solution to eachwell. The plates were incubated at 37° C. in a 5% CO₂ humidified chamberfor 90 minutes and read using a spectrophotometer at 490 (Spectra Max340, Molecular Devices, Sunnyvale, Calif.).

OX40R Antibody Selectivity Assay (ELISA):

Maxisorb 96-well plates (Nunc, Roskilde, Denmark) were coated with 100μl of human TNFα receptor family member fusion proteins at 0.25 μg/mldiluted in BupH™ Carbonate buffer, pH 9.4 (Pierce, Rockford, Ill.) andincubated overnight at 4° C. The selectivity receptor fusion proteinstested included CD40-Ig (Alexis Biochemicals, San Diego, Calif.),CD137-Ig (R&D Systems, Minneapolis, Minn.) and CD271-Ig (AlexisBiochemicals, San Diego, Calif.). Also included as the positive controlwith each assay was the OX40-Ig fusion protein (in-house construct,Bioexpress, 97/2117). Plates were then washed three times with washbuffer containing 0.05% Tween 20 (Sigma, St Louis, Mo.) diluted in PBSand blocked with 300 μl of 0.5% BSA (Sigma, St Louis, Mo.) in PBS(Sigma, St Louis, Mo.) for 1 hour at RT °. Next, the plates were washedand 100 μl/well of anti-human OX40 reactive antibodies were added to theplates at various concentrations and incubated for 1 hour at RT °.Plates were thoroughly washed three times and OX40R antibody binding wasdetected with a horse radish peroxidase labeled anti-human kappa chainantibody (Bethyl Laboratories, Montgomery, Tex.) at 25 ng/ml for 1 hourat RT °. Plates were then washed three times which was followed by theaddition of 100 μl/well of 1-Step Turbo-TMB substrate (Pierce, Rockford,Ill.) for 30 minutes at RT °. The reaction was stopped by adding anequal volume of 2M H₂SO₄. Absorbance was read at 450 nm on a MolecularDevices Spectra Max 340 (Molecular Devices, Sunnyvale, Calif.).

Species Cross-Reactivity:

Cell Lines Expressing OX40R: The 300-19 cell line expressing either arecombinant human OX40-CD40 fusion construct corresponding to 1-235 ofOX40R (extracellular and transmembrane domain) and 216-278 of CD40(intracellular domain) or the entire cynomolgus OX40R protein.

Preparation of Human T Lymphocytes:

Human whole blood was collected into heparinized syringes (Baxter;Deerfield, Ill.) and then immediately transferred to Sigma Accuspintubes (Sigma, St. Louis, Mo.) for the isolation of peripheral bloodmononuclear cells (PBMC) as described by the manufacturer. The PBMC werewashed twice with DPBS and T lymphocytes were isolated using a T cellpurification column as described by the manufacturer (R & D Systems,Minneapolis, Minn.). Briefly, PBMCs were resuspended in 2 mls of columnbuffer and loaded into a pre-washed T cell isolation column. PBMCs wereincubated for 10 minutes at room temperature and T cells were elutedwith column buffer, washed one time and resuspended TCM at 2×10⁶/mlconsisting of RPMI 1640 (Sigma, St Louis, Mo.) supplemented with 10%fetal bovine serum (Hyclone, Logan, Utah) and L-glutamine (2 mM), Hepes(10 mM), penicillin (100 U/ml), streptomycin (50 ug/ml) (Gibco, GrandIsland, N.Y.). A 2 ml volume of T cells containing an anti-human CD28antibody at 1 ug/ml (clone 37407, R & D Systems, Minneapolis, Minn.) wasadded to the wells of a 24 well plate pre-coated with an anti-human CD3antibody clone UCTH1 (R & D Systems, Minneapolis, Minn.) at 5 μg/ml inPBS. T cell cultures were stimulated for 3 days prior to being testedfor human OX40 cross-reactivity by flow cytometry.

Preparation of Cynomolgus PBMCs:

Cynomolgus whole blood was obtained using heparinized vacutainer tubes(BD; Franklin Lakes, N.J.) and was diluted 1:4 in PBS. Diluted wholeblood was mixed and 15 mls was carefully layered over an equal volume ofHistopaque 1077 (Sigma, St Louis, Mo.). The tubes were spun at 1000×gfor 45 minutes at RT ° and the mononuclear PBMC interface was harvested,washed once in PBS and resuspended for 2 minutes at RT ° with ACK lysingbuffer (Biosource, Rockville, Md.) to remove any RBCs. After a PBS wash,the PBMCs were counted and readjusted to 1×10⁶/ml in tissue culturemedium (TCM). TCM consisted of RPMI 1640 (Sigma, St Louis, Mo.)supplemented with 10% fetal bovine serum (Hyclone, Logan, Utah) andL-glutamine (2 mM), Hepes (10 mM), penicillin (100 U/ml), streptomycin(50 ug/ml) purchased from Gibco (Grand Island, N.Y.). Next, 2 mls of thePBMC preparation containing an anti-human CD28 cross-reactive antibody(clone CD28.2, BD Biosciences, San Diego, Calif.) was added to the wellsof a 24 well plate (Costar, Corning, N.Y.) pre-coated with ananti-monkey CD3 antibody (clone FN18, Biosource, Camarillo, Calif.) at10 μg/ml in PBS. PBMC cultures were stimulated for 4 days prior to beingtested for human OX40 cross-reactivity by flow cytometry.

Preparation of Rabbit PBMCs:

Rabbit whole blood was drawn into heparinized vacutainer tubes (BD;Franklin Lakes, N.J.) and immediately diluted 1:3 with warm HBSS (Gibco,Grand Island, N.Y.). After mixing, 5 mls of the diluted blood wascarefully layered over and equal volume of Lympholyte-Rabbit (CedarlaneLaboratories, Westbury, N.Y.) and centrifuged for 30 minutes at 25° C.The PBMC interface was collected, washed twice with PBS and resuspendedto 2×10⁶/ml in TCM containing PHA at 10 ng/ml (Remel, Lenexa, Kans.).The cells were cultured for 24-48 hours.

Preparation of Canine PBMCs:

Canine whole blood was collected using heparinized vacutainer tubes (BD;Franklin Lakes, N.J.). Next, the blood was mixed with an equal volume ofwarm HBSS (Gibco, Grand Island, N.Y.). Four mls of diluted blood wasslowly layered over 3 mls of Lympholyte-M (Cedarlane Laboratories,Westbury, N.Y.) in a 15 ml conical tubes. The tubes were centrifuged for20 minutes at 800×g and the PBMC interface was collected, washed twicewith HBSS and resuspended in TCM at 2×10⁶/ml. PBMCs were added to thewells of a 24 well plate (2 ml/well) and the cells were stimulated with2 μg/ml of ConA (Sigma, St. Louis, Mo.) for 48 hours.

Preparation of Murine and Rat PBMCs:

Rat whole blood collected in heparinized syringes was diluted 1:3 inwarm HBSS. Next, 5 mls was carefully layered over an equal volume ofLympholyte-Rat (Cedarlane Laboratories, Westbury, N.Y.). The tubes werecentrifuged for 20 minutes at 1500 RPM. The PBMCs interface wascollected, washed twice and the cell pellet was re-adjusted to 2×10⁶/mlin TCM. Two mls of cells were added to each well of a 24 well plate andstimulated for 24-48 hours with PHA (Remel, Lenexa, Kans.) at 10 ng/mlprior to flow cytometry staining.

Flow Cytometry Staining for Species Cross-reactivity:

Stimulated mouse, rat, rabbit, dog and cynomolgus PBMCs and the 300-19cell line expressing the cynomolgus OX40 receptor were used to test forhuman OX40 antibody species cross-reactivity. Human OX40 expressingactivated T lymphocytes and OX40 transduced 300-19 cells were used aspositive controls. Cells (5.0×10⁵/tube) were washed once in cold washbuffer (PBS, 2% FBS and 0.02% sodium azide) and 100 μl/tube of AlexaFluor 647 conjugated control or OX40 reactive antibodies at 5 ug/ml wasadded to each tube. The antibodies were labeled using an Alex Fluor 647protein labeling kit as described by the manufacturer (Molecular Probes,Eugene, Oreg.). The cells were incubated in the dark with fluorochromeantibodies on ice for 30 minutes, washed three times and resuspended in0.5 ml wash buffer for analysis. Antibody staining was measured andanalyzed using a Becton Dickinson FACSCalibur and CellQuest software(San Jose, Calif.).

Luciferase Activity Assay:

293T cells containing the extracellular domain of OX40 and theintracellular domain of CD40 fused to a NfkB reporter containingluciferase were prepared. Cells were harvested, washed and resuspendedinto phenol red free complete medium (DMEM containing 10% fetal bovineserum, HEPES buffer, nonessential amino acids and L-glutamine) atdensity of 0.5×10⁶ cell/ml. 80 ul of cells were plated into each assaywell of a 96 well plate (PerkinElmer, parts number 6005680). Testantibodies were added to each well alone or in the presence of a crosslinking antibody Fab′ goat anti-human IgG (Jackson ImmunoResearch, WestGrove, Pa.). The plate was incubated overnight at 37 C. 100 ul ofluciferase (Promega, Bright-glo luciferassay system, Cat. #E2620) wasadded the next day and the amount of luciferase activity was measuredusing a syntillation counter (TopCount, Packard-NXT).

Human αCD3 IL-2 Assay:

Human whole blood was collected in heparinized (Baxter; Deerfield, Ill.)syringes, layered over Accuspin tubes (Sigma; St. Louis, Mo.) andcentrifuged for 15 minutes at 2000 rpm's. The buffy coat was collected,washed with PBS (Sigma, St. Louis, Mo.), and red blood cells lysed withwater. T cells were separated out by human CD3⁺ enrichment columns (R&D;Minneapolis, Minn.), counted and adjusted to 1×10⁶/ml in RPMI media(Gibco; Grand Island, N.Y.) containing: 10% fetal calf serum (Hyclone;Logan, Utah), 10 mM hepes, 1% penicillin-streptomycin, 2 mM L-glutamineand 0.1 mM non-essential amino acids (all Gibco). Concurrently, humananti-CD3ε clone #UCHT1 (R&D systems, Minneapolis, Minn.) was placed at2.5 μgs/ml in PBS into 24 well plates (Costar; Corning, N.Y.) andincubated for 2 hours at 37° C. The plates were washed 3× with PBS andthe following added to the wells: T cells at 1×10⁶/well, serialdilutions of OX40 antibodies (or IgG₂ KLH control) and F(ab′)₂ goatanti-human IgG Fcγ to cross link (added at 2.5 ug/mL). Supernatants werepulled at 48 and 72 hours and IL-2 levels were assessed by ELISA (R&D).

Cynomolgus αCD3 IL-2 Assay:

Cynomolgus monkey whole blood was collected in heparinized tubes (BD;Franklin Lakes, N.J.), diluted 1:4 in PBS, layered over Histopaque 1077(Sigma, St Louis, Mo.) and centrifuged for 45 minutes at 2200 rpm's. Thebuffy coat was collected, washed with PBS, and red blood cells lysedwith water. Cells were adjusted to 1×10⁶/ml and added to 24 well platesthat had been pre-coated for 2 hours with varying concentrations ofmonkey anti-CD3, clone FN-18 (Biosource; Camarillo, Calif.) at 37° C.Serial dilutions of OX40 antibody (or IgG₂ KLH control), as well asF(ab′)₂ goat anti-human IgG Fcγ at 2.5 ug/mL were added to the wells.Supernatants were collected at 24 and 48 hours and IL-2 levels wereassessed by ELISA (Biosource, Camarillo, Calif.).

Alloantigen Primed T Cells Assay:

Freshly isolated human T cells (see above) were incubated with mitomycinc treated allogeneic tumor cells (Raji) for 3-4 days. T cells were thenharvested, washed, and rested for 1 day in fresh media prior tostimulating with 11D4. The level of IL-2 was assessed 24 hours latter byELISA (R&D systems, Minneapolis, Minn.).

B. In Vivo Study Procedures

SCID-Beige Human Tumor Models Using Mice Engrafted with Human T cellsand Dendritic Cells:

SCID-beige mice (Taconic #CBSBG-MM) were acclimated for 5-7 days afterarrival prior to use. The following tumor cell lines were used: RAJI,ATCC #CCL-86; BT-474, ATCC #HTB-20; PC-3, ATCC#-1435; and LoVo, ATCC#CCL-229.

Purified T lymphocytes (T cells) and monocyte derived dendritic cellswere prepared from human blood as follows: Human mononuclear cells werecollected from heparinized blood using Sigma Accuspin Tubes #A7054.Cells were collected, placed in a T75 flask, and incubated for 3 hrs at37° C. in a humidified incubator under 5% CO₂. The non-adherent cellswere collected and saved (see below). The flask containing the adherentcells was incubated with 20 ml RPMI complete medium (containing 10%fetal calf serum) supplemented with IL-4 (R&D) at 10 ng/ml and GM-CSF(R&D) at 100 ng/ml. The culture was then incubated for 6-7 days at 37°C. in a humidified incubator under 5% CO₂. The non-adherent monocytederived dendritic cells were then collected by decanting and rinsingflask several times with RPMI complete medium.

The initial non-adherent mononuclear cells were used to purify T cellsvia high affinity negative selection using T cell enrichment columns(R&D) as per manufacturer's instructions. Purified T cells arecryo-preserved in Recovery-Cell Culture Medium at 10⁷/ml and stored inliquid Nitrogen until use. Tumor cells (1×10⁷) were injectedsubcutaneously (SC) with T cells (1×10⁶) and monocyte-derived dendriticcells (5×10⁵) from the same donor, at 0.2 mL/mouse. Tumor growth wasmonitored over time with calipers.

C. Results for Antibody 11D4

(1) In Vitro Studies:

Certain properties of antibody 11D4 from in vitro studies are summarizedin Table 3.

Antibody 11D4 Binds to the OX40R with High Affinity.

This was demonstrated by using an IgG1 fusion protein containing theextracellular domain of the OX40R and on whole cells (OX40R+ transfectedcells and activated primary T cells). In examples using the IgG1 fusionprotein, 11D4 bound to the extracellular domain of the OX40R with anEC₅₀ of 0.5+/−0.18 μg/mL (3.5 nM). This binding was confirmed on 300-19pre-B cells expressing the full length extracellular domain of the OX40R(no binding was observed on parental 300-19 cells). The EC₅₀ for bindingto OX40R transfected cells was 0.2+/−0.16 μg/mL (1.7 nM). In order toconfirm that binding was observed on primary T cells, peripheral blood Tcells were isolated from multiple human donors and stimulated withanti-CD3 and anti-CD28 for 2 days to upregulate the expression of theOX40R. Saturation binding data on these T cells indicated that 11D4binds with an EC₅₀ of 0.6+/−1.0 μg/mL (4.0 nM, N=17 donors). These datademonstrate that 11D4 avidly binds to the OX40R.

In order to further characterize this binding, data was collected toassess the region on the extracellular domain of the OX40R where 11D4interacts and to also determine whether the receptor was internalizedfollowing binding. Competition binding data to the OX40R IgG1 fusionprotein indicated that 11D4 competes for binding with OX40 ligandexpressing cells providing evidence that 11D4 interacts at the ligandbinding region of the receptor. In addition, 11D4 does not cross-competewith two commercially available OX40R antibodies, BerAct35 and L106, forbinding to T cells as assessed by FACS analysis. FACS analysis usingnon-competing detection antibodies indicated that the OX40R was notinternalized following the pre-incubation of primary, activated T cellswith 11D4 for 30 minutes. Its binding affinity, determined by Biacoreanalysis using the OX40R extracellular domain fusion protein as theimmobilized ligand, indicated that the equilibrium dissociation constant(KD) of 11D4 for binding was 0.48 nM. These analyses also estimated theoff rate constant (kd) of 11D4 to be 5.72 E-05 l/s. Therefore, 11D4binds with high affinity to the ligand binding region of the OX40R, hasa slow off-rate constant, and does not internalize the receptorfollowing binding.

Antibody 11D4 Selectively Binds to the OX40R.

The selectivity of 11D4 for the OX40R was assessed against other membersof the TNFR superfamily using data related to IgG1 fusion proteinconstructs containing the respective extracellular domain of the relatedreceptor. These receptors included the CD40 receptor, 4-1BB receptor(CD137) and the nerve growth factor receptor (CD271). In all cases, nosignificant binding was observed at concentrations up to 100 μg/mL (700nM) on these receptors. When compared to binding observed to the OX40Rfusion protein (EC₅₀=0.5 ug/ml), these data demonstrate that 11D4is >100-fold selective for the OX40R vs other related family memberstested. (See FIGS. 1a and 1b ).

Functional Activity of Antibody 11D4:

The functional activity of 11D4 was demonstrated on both OX40R+transfected cells and on primary T cells. In these assays, 11D4demonstrated agonist activity when added to cells with or without asecondary antibody, F(ab′)₂ goat anti-human IgG Fcγ.

In the first set of experiments, 11D4 was assessed for agonist activityusing 293 cells transfected with the extracellular and transmembranedomain of the OX40R fused to the intracellular domain of CD40 with anNFkB luciferase reporter. In this assay, 11D4 enhanced signaling throughthe OX40R with a mean EC₅₀ of 0.33 μg/mL (2.2 nM, N=4). A representativeconcentration-response curve for the induction of luciferase by 11D4 isshown in FIG. 2. In the absence of the F(ab′)₂ secondary antibody, themagnitude of luciferase activity was reduced 4-fold along with the EC₅₀.

As further evidence for the agonist activity of 11D4, antigen-specific Tcells were generated. Freshly isolated human T cells were incubated withmitomycin c treated allogeneic tumor cells (Raji) for 3-4 days. T cellswere then harvested, washed, and rested for 1 day in fresh media priorto stimulating with 11D4. FACS analysis indicated a high level of OX40Rexpression on these cells even after resting. 11D4 induced high levelsof IL-2 by these cells, in some cases exceeding 100 ng/mL (FIG. 3). Theaverage EC₅₀ for this response from 2 separate examples was0.008+/−0.006 μg/mL. In the absence of 11D4, only minimal levels of IL-2were secreted by these cells.

11D4 also enhances the IL-2 production by the primary human T cellsstimulated by anti-CD3. Although the signal to noise ratio in this assaywas low in some assays due to the induction of IL-2 by anti-CD3 alone,11D4 enhanced IL-2 production when added with F(ab′)₂ goat anti-humanIgG Fcγ. No activity was observed for 11D4 on freshly isolated T cellsin the absence of anti-CD3. The magnitude of IL-2 augmentation by 11D4ranged from 2.3 to 57-fold vs. anti-CD3 alone depending on the donor andthe amount of IL-2 generated by anti-CD3. The effect of 11D4 on IL-2production by primary human T cells stimulated with 2.5 μg/mL anti-CD3is represented in FIG. 4 (using an 8 point concentration curve with 1:3dilutions). The average EC₅₀ calculated from those data which used8-point concentration response curves was 0.042+/−0.01 μg/mL (see Table4).

Functional activity of 11D4 on IL-2 production was also assayed usingmonkey cells stimulated with anti-CD3 and 11D4 (along with F(ab′)₂secondary antibody). Results are represented in FIG. 5 and Table 5.These data indicated that the EC₅₀ for 11D4 was similar between monkeyand human cells (0.022 vs 0.042 μg/mL for human cells), but themagnitude of IL-2 induced above that of anti-CD3 alone was significantlyless using Cynomolgus T cells (approx. 35-fold, 5762+/−4748 pg/mL IL-2for human cells (N=21) vs 261+/−294 pg/mL IL-2 for monkey cells (N=9).

Species Cross-Reactivity:

11D4 was assessed for its ability to bind to T cells from multiplespecies. T cells were isolated from mouse, rat, rabbit, dog, and monkeyand activated with either anti-CD3 plus anti-CD28 or mitogen. No bindingwas observed to mouse, rat, rabbit or dog cells as indicated by FACSanalysis. The lack of binding to mouse OX40R was also confirmed by ELISAusing a commercially available fusion protein containing theextracellular domain of the murine OX40R. In contrast, 11D4 binds toCynomolgus monkey T cells as determined in a saturation binding assay byFACS. The range of EC₅₀ values obtained using different monkeys is shownin FIG. 6. For comparison, the range of EC₅₀ values obtained using humancells is shown in FIG. 7. Although variable, the range of EC₅₀ valueswas similar between monkey and human cells (mean values are 0.354 μg/mLfor monkey vs 0.566 μg/mL for human cells).

(2) In Vivo Studies:

The lack of 11D4 cross-reactivity with the murine OX40R required thedevelopment of a xenogenic tumor model using Severe CombinedImmunodeficient (SCID) beige mice. SCID-beige mice lack murine T and Blymphocytes and NK cells making them ideal recipients for theengraftment of human immune cells and the growth of human tumors. Fourtumor cell lines representing diverse tumor types were tested in this invivo model. None of the tumor lines expressed OX40R. In all cases, tumorcells (1×10⁷) were injected subcutaneously (SC) with T cells (1×10⁶) andmonocyte derived dendritic cells (5×10⁵) from the same donor. 11D4administered by intraperitoneal (IP) injection inhibited tumor growth upto 98% in these models as summarized in Table 6. The IP route ofadministration was chosen for 11D4 due to its ease of administration andrapid dissemination into the peripheral blood.

Efficacy of 11D4 Against a B Cell Lymphoma in SCID-Beige Mice:

SCID-beige mice were injected SC with the Burkitt's B cell lymphoma,Raji, together with human T cells and monocyte-derived dendritic cells.Mice received a single IP injection of either 11D4 or an isotype controlantibody (IgG2 anti-KLH) at the time of tumor injection. As shown inFIG. 8, 11D4 decreased the rate of tumor growth in treated animals. Thetumor size in each individual animal (N=10) on day 21 after challenge isshown in FIG. 9, illustrating 64% inhibition in tumor growth by a doselevel of 10 mg/kg. No activity was observed in the absence of T cellsand dendritic cells.

Efficacy of 11D4 in a Prostate Tumor Model:

SCID-beige mice were injected SC with the prostate adenocarcinoma PC-3together with human T cells and monocyte-derived dendritic cells. Micereceived a single IP injection of either 11D4 or an isotype controlantibody (IgG2 anti-KLH) at the time of tumor injection. The results,which are represented in FIG. 10, show that 11D4 treatment resulted in adose-dependent inhibition of tumor growth. The tumor size in eachindividual animal (N=10) from this study on day 27 after challenge isshown in FIG. 11, illustrating a 70% inhibition in tumor growth whenanimals were administered a single injection of 1.0 mg/kg 11D4, and 90%inhibition at a dose of 10 mg/kg. The plasma levels of 11D4 determinedon day 27 in these animals were 6.2 μg/mL at the 1.0 mg/kg dose level.

Efficacy of 11D4 in a Colon Carcinoma Tumor Model:

SCID-beige mice were injected SC with the colorectal adenocarcinoma LoVotogether with human T lymphocytes and autologous monocyte-deriveddendritic cells. Mice received a single IP injection of either 11D4 or acontrol antibody (IgG2 anti-KLH) at the time of tumor injection. Theresults, which are represented in FIG. 12, show that 11D4 dosedependency decreased tumor growth in these animals. The tumor size ineach individual animal (N=10) from this study on day 27 after challengeis shown in FIG. 13, illustrating a 64% inhibition in tumor growth usinga single dose of 1.0 mg/kg and a 87% inhibition of tumor growth at adose level of 10.0 mg/kg.

Efficacy of 11D4 in a Mammary Carcinoma Tumor Model

SCID-beige mice were injected SC with the mammary carcinoma BT474together with human T lymphocytes and autologous monocyte-deriveddendritic cells. Mice received two injections (IP) of either 11D4 or acontrol antibody (IgG2 anti-KLH) at the time of tumor injection andagain 30 days later. The results, which are represented in FIG. 14, showthat 11D4 decreased tumor growth in these animals. The tumor size ineach individual animal (N=10) from this study on day 85 after challengeis shown in FIG. 15 illustrating a 98% inhibition in tumor growth at adose level of 10.0 mg/kg and 85% inhibition at a dose of 1 mg/kg.

D. Results for Antibody 18D8

(1) In Vitro Studies:

Results from in vitro studies for antibody 18D8 are summarized in Table7.

Effect of antibody 18D8 on anti-CD3 induced IL-2 production by primaryhuman T cells from different donors are also shown in Table 8.

(2) In Vivo Studies:

Efficacy of 18D8 Against B Cell Lymphoma in a SCID-Beige Mice Model

SCID-beige mice were injected SC with the Burkitt's B cell lymphoma,Raji, together with human T lymphocytes and autologous monocyte-deriveddendritic cells. Mice received a single IP injection of either 18D8 oran isotype control antibody (IgG2 anti-KLH) at the time of tumorinjection. Ten animals per group were used in each study. The resultsfrom two studies are presented in Table 9. The results show that 18D8produced significant anti-tumor efficacy at the doses of 1.0 mg/kg and10 mg/kg. No activity was observed in the absence of T cells anddendritic cells, suggesting that this anti-tumor effect may be immunemediated.

Efficacy of 18D8 Against Prostate Tumor in a SCID-Beige Mice Model

SCID-beige mice were injected SC with the prostate adenocarcinoma PC-3together with human T cells and autologous monocyte-derived dendriticcells. Mice received a single IP injection of either 18D8 or an isotypecontrol antibody (IgG2 anti-KLH) at the time of tumor injection. Tenanimals per group were used in the study. The results are presented inTable 9. The results show that 18D8 treatment resulted in a 42%, 90%,and 88% inhibition of tumor growth at the doses of 0.1 mg/kg, 1.0 mg/kg,and 10 mg/kg, respectively.

DEPOSIT INFORMATION

Applicants have deposited a culture of E. coli DHα5 containing plasmidthat encodes the heavy chain of antibody 11D4 and a culture of E. coliDHα5 containing plasmid that encodes the light chain of antibody 11D4 inthe American Type Culture collections (ATCC), 10801 UniversityBoulevard, Manassas, Va. 20110-2209, on Jul. 10, 2007, which have beenassigned deposit numbers PTA-8524 and PTA-8525, respectively. Thesedeposits were made under the provisions of the Budapest Treaty on theInternational Recognition of the Deposit of Microorganisms for thePurpose of Patent Procedure and the Regulations thereunder (BudapestTreaty). These deposits will be maintained without restriction in theATCC depository for a period of 30 years, or 5 years after the mostrecent request, or for the effective life of the patent, whichever islonger, and will be replaced if the deposits become non-viable duringthat period. Availability of the deposited materials is not to beconstrued as a license to practice the invention in contravention of therights granted under the authority of any government in accordance withits patent laws.

All references cited in this specification, including, withoutlimitation, all papers, publications, patents, patent applications,books, journal articles, are hereby incorporated by reference into thisspecification in their entireties. The discussion of the referencesherein is intended to merely summarize the assertions made by theirauthors and no admission is made that any reference constitutes priorart.

Although the foregoing invention has been described in some detail byway of illustrations and examples for purposes of clarity ofunderstanding, it will be readily apparent to those of ordinary skill inthe art in light of the teachings herein that certain changes andmodifications may be made to the invention without departing from thespirit or scope of the appendant claims.

TABLE 1 Sequence Identifiers for Antibodies 11D4 and 18D8 SEQ ID NO:Antibody Sequence 1 11D4 V_(H) CDR1 Amino Acid 2 11D4 V_(H) CDR2 AminoAcid 3 11D4 V_(H) CDR3 Amino Acid 4 11D4 V_(L) CDR1 Amino Acid 5 11D4V_(L) CDR2 Amino Acid 6 11D4 V_(L) CDR3 Amino Acid 7 11D4 V_(H) AminoAcid 8 11D4 V_(L) Amino Acid 9 11D4 Heavy Chain Amino Acid 10 11D4 LightChain Amino Acid 11 11D4 V_(H) Nucleic Acid 12 11D4 V_(L) Nucleic Acid13 18D8 V_(H) CDR1 Amino Acid 14 18D8 V_(H) CDR2 Amino Acid 15 18D8V_(H) CDR3 Amino Acid 16 18D8 V_(L) CDR1 Amino Acid 17 18D8 V_(L) CDR2Amino Acid 18 18D8 V_(L) CDR3 Amino Acid 19 18D8 V_(H) Amino Acid 2018D8 V_(L) Amino Acid 21 18D8 Heavy Chain Amino Acid 22 18D8 Light ChainAmino Acid 23 18D8 V_(H) Nucleic Acid 24 18D8 V_(L) Nucleic Acid

TABLE 2A Amino Acid Sequences for Antibody 11D4SEQUENCE (Variable region in upper case, constant region in DESCRIPTIONlower case, CDRs underlined) Heavy ChianEVQLVESGGGLVQPGGSLRLSCAASGFTFSSY SMNWVRQAPGKGLEWVSYISSSSSTIDYADSVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYC ARESGWYLFDYWGQGTLVTVSSastkgpsvfplapcsrstsestaalgclvkdyfpepvtvswn sgaltsgvhtfpavlqssglyslssvvtvpssnfgtqtytcnvdhkpsntkvdktverkccvec ppcpappvagpsvflfppkpkdtlmisrtpevtcvvvdvshedpevqfnwyvdgvevhnaktkp reeqfnstfrvvsvltvvhqdwlngkeykckvsnkglpapiektisktkgqprepqvytlppsr eemtknqvsltclvkgfypsdiavewesngqpennykttppmldsdgsfflyskltvdksrwqq gnvfscsvmhealhnhytqkslslspgkLight Chain DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSG SGSGTDFTLTISSLQPEDFATYYCQQYNSYPPTFGGGTKVEIKrtvaapsvfifppsdeqlksg tasvvcllnnfypreakvqwkvdnalqsgnsqesvteqdskdstyslsstltlskadyekhkvy acevthqglsspvtksfnrgec

TABLE 2B Amino Acid Sequences for Antibody 18D8SEQUENCE (Variable region in upper case, constant region in DESCRIPTIONlower case, CDRs underlined) Heavy ChianEVQLVESGGGLVQPGRSLRLSCAASGFTFDDY AMHWVRQAPGKGLEWVSGISWNSGSIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYC AKDQSTADYYFYYGMDVWGQGTTVTVSSastkgpsvfplapcsrstsestaalgclvkdyfpep vtvswnsgaltsgvhtfpavlqssglyslssvvtvpssnfgtqtytcnvdhkpsntkvdktver kccvecppcpappvagpsvflfppkpkdtlmisrtpevtcvvvdvshedpevqfnwyvdgvevh naktkpreeqfnstfrvvsvltvvhqdwlngkeykckvsnkglpapiektisktkgqprepqvy tlppsreemtknqvsltclvkgfypsdiavewesngqpennykttppmldsdgsfflyskltvd ksrwqqgnvfscsvmhealhnhytqkslslsp gkLight Chain EIVVTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSG SGSGTDFTLTISSLEPEDFAVYYCQQRSNWPTFGQGTKVEIKrtvaapsvfifppsdeqlksgt asvvcllnnfypreakvqwkvdnalqsgnaqesvteqdskdstyslsstltlskadyekhkvya cevthqglsspvtksfnrgec

TABLE 3 Summary of Certain in vitro Properties of Antibody 11D4Parameter Activity μg/ml (nM) Affinity for OX40R: (Biacore) K_(D) 0.070.48 Off rate (kd) 5.7E−05 1/s Binding to OX40R: Fusion proteinextracellular domain 0.5 +/− 0.18 3.50 Saturation binding (EC₅₀):CD3/CD28 stimulated T cells 0.6 +/− 1.00 4.00 (N = 17) OX40R + 300-19cells (N = 5) 0.2 +/− 0.16 1.70 In vitro stimulation of OX40R + 0.33 +/−0.22  2.20 transfected cells (luciferase) (EC₅₀; N = 4) Augmentation ofT cell activity: CD3 induced IL-2 production 0.042 +/− 0.01  0.30 (N =12) Stimulation of IL-2 production by 0.008 +/− 0.006  0.04antigen-primed cells (N = 2) Selectivity (binding) >100 μg/mL    >700.00(CD40, CD137, CD271) Values represent the mean +/− one SD

TABLE 4 Effect of Antibody 11D4 on Anti-CD3 Induced IL-2 Production byPrimary Human T Cells EC₅₀ Max IL-2 ECmax Stimulation (μg/mL) (pg/mL)(μg/mL) Index Donor 0.008 4831 0.05 3.8 1 0.011 5450 0.05 2.6 2 0.0146571 0.5  2.3 3 0.014 7271 0.05 5.9 4 0.011 6313 0.05 9.1 5 ND ND ND 7.06 0.010 1006 0.05 4.8 7 ND ND ND 5.9 8 ND ND ND 25.4 9 ND ND ND 57.0 10ND ND ND 8.3 11 ND ND ND 5.1 12 ND ND ND 2.7 13 ND ND ND 4.6 14 0.0144687 0.05 6.0 15 0.014 3012 0.05 35.2 16 ND ND ND 21.4 17 0.029 2796 0.125 3.8 18 0.052 1718  0.125 5.5 19 0.020 14190  0.56 16.8 20 0.0681611 1.67 7.9 21 Max IL-2: Amount of IL-2 produced with 11D4 at theECmax over anti-CD3 alone ECmax: Concentration of 11D4 producing themaximum level of IL-2 over anti-CD3 alone Stimulation Index: Ratio ofthe maximum level of IL-2 produced with 11D4 vs the amount of IL-2produced with anti-CD3 alone ND = not determined. Values for the lastfour donors (18-21) are from dose response curve done in 8-point 1:3 or1:4 dilutions; all other values represent log dilution curves. The EC₅₀from the 1:3 and 1:4 concentration curves was 0.042 +/− 0.01 ug/mL, N =4.

TABLE 5 Effect of Antibody 11D4 on Anti-CD3 Induced IL-2 Production byCynomolgus T Cells. Max IL-2 EC₅₀ Induced ECmax Stimulation (μg/mL)(pg/mL) (μg/mL) index Donor 0.007 376 0.05  3.5x 32750 0.002 116 0.05 2.2x 2325 ND ND ND 2.0x 32405 0.007 167 0.05  34.4 32081 0.011 978 0.0055.6x 32842 ND ND ND 6.3x 2325 0.008  40 0.021 2.7x 33081 0.031 168 0.0625.0x 33080 0.028 128 0.062 3.8x 33062 Max IL-2: Amount of IL-2 producedwith 11D4 at ECmax over anti-CD3 alone ECmax: Concentration of 11D4producing the maximum level of IL-2 over anti-CD3 alone StimulationIndex: Ratio of the maximum IL-2 produced with 11D4 over the amountproduced with anti-CD3 alone ND = not determined

Values for the last three donors (33081, 33080, and 33062) in Table 6are from dose-response curve done in 8-point 1:3 dilutions. All othervalues represent log dilution curves. The EC₅₀ derived from those curvesusing 1:3 dilutions was 0.022+/−0.01; N=3.

TABLE 6 Human Tumor Growth Inhibition by Antibody 11D4 In SCID- beigeMice Engrafted with Human T cells and Dendritic Cells Dosing Study 101.0 0.1 0.01 Tumor Type with 11D4 Duration mg/kg mg/kg mg/kg mg/kg Raji:B cell Day 1 21 days 64% 42% 27% nd lymphoma Raji: B cell Day 1 21 daysnd 75% 42% 8% lymphoma Lovo: colon Day 1 25 days 76% 44% 20% ndcarcinoma Lovo: colon Day 1 25 days 87% 64% 15% nd carcinoma PC3:prostate Day 1 27 days 90% 77% 45% nd PC3: prostate Day 1 27 days 90%70% 50% nd BT474: Day 1 85 days 98% 85% 46% nd breast and 30 Values = %inhibition of tumor growth determined at study termination nd = notdetected

TABLE 7 Results of in vitro Studies with Antibody 18D8 ActivityParameter μg/ml (nM) Affinity for OX40R (Biacore): K_(D) 0.49 3.38 Offrate (kd) 2.9E−04 1/s Binding to OX40R (EC₅₀): Fusion proteinextracellular domain 0.034 +/− 0.01 0.23 Saturation binding: CD3/CD28stimulated T cells (N = 4)  1.06 +/− 0.51 7.30 OX40R + 300-19 cells (N =2)  0.24 +/− 0.09 1.66 Augmentation of T Cell Activity (EC₅₀): CD3induced IL-2 production (N = 4) 0.049 +/− 0.06 0.33 Stimulation of IL-2production by 0.014 +/− 0   0.10 antigen-primed cells (N = 1)Selectivity (Binding to CD40, CD137, >100 μg/mL   >700.00 CD271): Valuesfor activity expressed in μg/ml represent the mean +/− one SD.

TABLE 8 Effect of Antibody 18D8 on Anti-CD3 Induced IL-2 Production byPrimary Human T Cells. EC₅₀ Max IL-2 ECmax Stimulation (μg/mL) (pg/mL)(μg/mL) Index Donor 0.013 1120 0.05 13.7 LC 0.024 4334 0.5 5.1 TH 0.0242280 0.5 5.4 KO 0.135 1356 0.5 2.4 RN Max IL-2: Amount of IL-2 producedwith 18D8 at the ECmax over anti-CD3 alone. ECmax: Concentration of 18D8producing the maximum level of IL-2 over anti-CD3 alone. StimulationIndex: Ratio of the maximum IL-2 produced with 18D8 over the amountproduced with anti-CD3 alone. Values represent log dilution curves.

TABLE 9 Inhibition of Human Tumor Growth by Antibody 18D8 in SCID-beigeMice Dosing Dose Level of 18D8 with Study (mg/kg) Tumor Type 18D8Duration 10 1.0 0.1 0.01 Raji: B cell lymphoma Day 1 23 days 73% 73% 11%nd Raji: B cell lymphoma Day 1 24 days 54% 59% nd nd PC3: prostate Day 124 days 88% 90% 42% nd Values = % inhibition of tumor growth determinedat study termination nd = not determined

We claim:
 1. An isolated nucleic acid molecule comprising a nucleic acidsequence that encodes a heavy chain or an antigen-binding fragmentthereof, and a light chain or an antigen-binding fragment thereof of anantibody that specifically binds OX40R, wherein said antibody comprises:(a) a heavy chain variable region (V_(H)) comprising: a V_(H) CDR1region comprising an amino acid sequence of SEQ ID NO: 13, a V_(H) CDR2region comprising an amino acid sequence of SEQ ID NO: 14, and a V_(H)CDR3 region comprising an amino acid sequence of SEQ ID NO: 15; and alight chain variable region (V_(L)) comprising: a V_(L) CDR1 regioncomprising an amino acid sequence of SEQ ID NO: 16, a V_(L) CDR2 regioncomprising an amino acid sequence of SEQ ID NO: 17, and a V_(L) CDR3region comprising an amino acid sequence of SEQ ID NO: 18; (b) a heavychain variable region comprising an amino acid sequence of SEQ ID NO:19; and a light chain variable region comprising an amino acid sequenceof SEQ ID NO: 20; or (c) a heavy chain comprising an amino acid sequenceof SEQ ID NO: 21; and a light chain comprising an amino acid sequence ofSEQ ID NO:
 22. 2. A vector comprising the isolated nucleic acid moleculeaccording to claim
 1. 3. The vector according to claim 2, furthercomprising an expression control sequence operably linked to saidisolated nucleic acid molecule.
 4. The vector according to claim 2,wherein said vector is selected from the group consisting of a plasmidexpression vector, a retrovirus expression vector, an adenovirusexpression vector, and an adeno-associated virus expression vector.
 5. Ahost cell comprising said vector according to claim
 2. 6. The host cellaccording to claim 5, wherein said host cell is selected from the groupconsisting of a mammalian cell, an insect cell, a plant cell, abacterial cell, and a yeast cell.
 7. A method for producing a monoclonalantibody or an antigen-binding fragment thereof that specifically bindshuman OX40R, comprising culturing said host cell according to claim 5under suitable conditions and recovering said monoclonal antibody orsaid antigen-binding fragment.
 8. An isolated nucleic acid moleculecomprising: (a) a nucleic acid sequence of SEQ ID NO: 23; (b) a nucleicacid sequence of SEQ ID NO: 24; or (c) a nucleic acid sequence of (a)and a nucleic acid sequence of (b).
 9. A vector comprising the isolatednucleic acid molecule according to claim
 8. 10. The vector according toclaim 9, further comprising an expression control sequence operablylinked to said isolated nucleic acid molecule.
 11. The vector accordingto claim 9, wherein said vector is selected from the group consisting ofa plasmid expression vector, a retrovirus expression vector, anadenovirus expression vector, and an adeno-associated virus expressionvector.
 12. A host cell comprising said vector according to claim
 9. 13.The host cell according to claim 12, wherein said host cell is selectedfrom the group consisting of a mammalian cell, an insect cell, a plantcell, a bacterial cell, and a yeast cell.
 14. A method for producing amonoclonal antibody or an antigen-binding fragment thereof thatspecifically binds human OX40R, comprising culturing said host cellaccording to claim 12 under suitable conditions and recovering saidmonoclonal antibody or said antigen-binding fragment.
 15. An isolatednucleic acid molecule comprising a nucleic acid sequence encoding theamino acid sequences of SEQ ID NOs: 13, 14, and
 15. 16. The isolatednucleic acid molecule according to claim 15, wherein said nucleic acidsequence further encodes the amino acid sequences of SEQ ID NOs: 16, 17,and 18.